WO2012025759A2

WO2012025759A2 - Method

Info

Publication number: WO2012025759A2
Application number: PCT/GB2011/051608
Authority: WO
Inventors: Peter Beverley; Elma Tchilian; Lian Ni Lee
Original assignee: Isis Innovation Limited
Priority date: 2010-08-26
Filing date: 2011-08-25
Publication date: 2012-03-01
Also published as: WO2012025759A3

Abstract

The invention relates to methods for determining the effectiveness of pulmonary immunisation of a subject by measuring CXCR6 or CXCL16. The invention also relates to methods for diagnosing a pulmonary infection in a subject or determining the effectiveness of a treatment of a pulmonary infection by measuring CXCR6 or CXCL16. The invention also relates to using CXCL16 as an adjuvant to increase the mononuclear cell response to an antigen.

Description

METHOD

Field of the Invention

The invention relates to methods for determining the effectiveness of pulmonary

immunisation of a subject by measuring CXCR6 or CXCL16. The invention also relates to methods for diagnosing a pulmonary infection in a subject or determining the effectiveness of a treatment of a pulmonary infection in a subject by measuring CXCR6 or CXCL16. The invention also relates to using CXCL16 as an adjuvant to increase the mononuclear cell response to an antigen.

Background to the Invention

In localized infections, targeting of the appropriate immune response to the correct location is essential to pathogen clearance. Pulmonary tuberculosis (TB) is largely an infection localized to the lung. In susceptible individuals, inhalation of a low number of bacilli in air droplets initiates infection. Unlike other infections, adaptive immunity is delayed as M. tuberculosis has to be transported to pulmonary lymph nodes (LN), which takes between 8-11 days, in order to prime effector T cells which then traffic back to lungs (Reiley et al., Proceedings of the National Academy of Sciences of the United States of America 2008, 105(31): 10961-10966). This delay allows M.

tuberculosis to steadily increase in lungs during this time. A similar delay in recruitment of antigen specific cells in occurs even in BCG-immunised individuals, where control is not established until at least 21 days post-infection (Winslow et al., Immunological reviews 2008, 225:284-299).

In a model of intranasal immunisation using the replication-deficient recombinant adenovirus Ad hu5 serotype expressing the 85A antigen from M. tuberculosis, control is effected early, within the first 7 days partly as immunisation results in the establishment of resident antigen-specific CD8 T cells in the lungs (Ronan EO et al., PloS one 2009, 4(12):e8235). By contrast, immunisation with the same vector through the intradermal (i.d.) route offers no protection against experimental aerosol TB challenge in mice (Forbes EK et al, J Immunol 2008, 181(7):4955-4964).

Trafficking of cells into tissues is mediated by chemokines, small secreted proteins which act as ligands of seven-transmembrane G protein-coupled receptors. Their presence on the cell surface allow these cells to be directed to relevant tissue through the establishment of a chemokine gradient. CXCR6 is a receptor which was initially described as a co-receptor for HIV (Alkhatib et al., Nature 1997, 388(6639):238), and then was subsequently found to allow T cells to migrate into tissues (Kim et al, The Journal of clinical investigation 2001, 107(5):595-601). Expression of CXCR6 has been detected on NK cells, NKT, DCs, and activated T cells (Unutmaz et al., J Immunol 2000,

165(6):3284-3292; and Motsinger et al., The Journal of experimental medicine 2002, 195(7):869- 879). It has been described as a marker for effector T cells in both humans and mice, which is controlled by interferon gamma (IFNy) (Calabresi et al., Journal of neuroimmunology 2002, 127(1- 2):96-105). CXCR6 positive T cells have been found in liver and help define the phenotype of a subset of CD8⁺ T cells during Hepatitis C infection (Northfield et al., Hepatology (Baltimore, Md 2008, 47(2):396-406). In cancer, it plays a role in regulating anti-tumour activity by recruiting i KT and tumour specific activated CD8 T cells to the tumour site, helping limit metastases (Cullen et al., J Immunol 2009, 183(9):5807-5815). Conversely, as a result of its ability to promote homing of lymphocytes to extralymphoid tissue, CXCR6 has been implicated in a number of T-cell mediated pathologies: it is reported to have a proatherosclerosis role by promoting homing and accumulation of lymphocytes to the aortic wall (Galkina et al., Circulation 2007, 116(16): 1801-1811), exacerbation of arthritis (Nanki et al, Arthritis and rheumatism 2005, 52(10):3004-3014), while in humans, constitutive expression of its only known ligand, CXCL16, in lung epithelia may be responsible for the long term retention of CD3⁺ T cells in lungs (Day et al., Experimental lung research 2009,

35(4):272-283) and may also play a role in the exacerbation of chronic obstructive pulmonary disease (Freeman et al., The American journal of pathology 2007, 171(3):767-776).

In lungs, CXCR6⁺ T cells have been found in biopsies of 'normal' lung tissue collected from lung cancer patients (Morgan et al, Immunobiology 2008, 213(7):599-608), during allergen-induced lung inflammation (Latta et al, Immunology 2007, 121(4):555-564) and are thought to exacerbate COPD (Freeman et al, The American journal of pathology 2007, 171(3):767-776) and sarcoidosis (Agostini et al, American journal of respiratory and critical care medicine 2005, 172(10): 1290- 1298). CXCR6 CD8 T cells were detected in lungs 7 days after immunisation with rotavirus vaccine by the intranasal (i.n.) but not via intraperitoneal (i.p.) or intramuscular (i.m.) routes (Jiang et al, Journal of virology 2008, 82(14):6812-6819). In a study describing the efficacy of a-GalCer as an adjuvant for influenza virus HA vaccine (Kamijuku et al, Mucosal immunology 2008, 1(3):208-218), it was found that i.n. but not i.m immunisation of H.A+a-GalCer resulted in cross-protection against heterosubtypic virus in a mouse model (B6 mice). The mechanism behind cross protection was found to be the high levels of secretary IgA produced in nasal secretions as a result of large influx of NKT cells into the NALT in response to i.n. immunisation. NKT cells were previously reported to increase IgA production by activating B-cells and antibody production and this study found that it was mediated in an IL-4 dependent manner. NKT cells express CXCR and this study found that ablation of CXCL16 prevented migration of NKT cells to the NALT and thus i.n. immunised CXCL16 K/O mice did not survive lethal pneumonia challenge. Therefore cross protection in this model is dependent on CXCR6-CXCL16 axis.

CXCL16 is the only known ligand for CXCR6 (Matloubian et al., Nature immunology 2000, l(4):298-304). It is a transmembrane chemokine, typically produced by immune cells, astrocytes, keratinocytes, fibroblasts and various tumours. Alternatively spliced form of CXCL16 is secreted (van der Voort et al, Journal of leukocyte biology, 87(6): 1029-1039).

Summary of the Invention

The inventors have surprisingly shown that CXCR6 is able to serve as a marker for successful intranasal (i.n.) immunisation. They found that i.n. immunisation with replication deficient adenoviruses (Ad85A) or subunit protein vaccines induces a long lived lung population of CXCR6⁺ effector CD8 T cells which are antigen specific and concentrated in the bronchoalveolar lavage (BAL). The inventors have also detected an increase in the serum level of CXCL16, the ligand for CXCR6, after an immunisation procedure that induces a protective immune response.

Furthermore, the inventors have surprisingly found that, in an immunisation regime which does not result in establishment of CXCR6⁺ lung T cells, co-delivery of its ligand CXCL16 with antigen is still able to mediate migration of large numbers of antigen- specific CD 8 T cells into the lumen of the lung airways.

The invention therefore provides a test for successful immunisation of the lung. This involves either demonstrating CXCR6 positive T cells in the lung or a rise in serum CXCL16 following immunisation. It may also involve demonstrating production of CXCL16 by lung mononuclear cells.

The invention also provides methods for diagnosing lung infections by demonstrating

CXCR6 positive T cells in the lung, production of CXCL16 by lung mononuclear cells or a rise in serum CXCL16. The invention further provides the use of the combination of CXCL16 and antigen for immunisation or immunotherapy. Administration of CXCL16 with an antigen into the lungs of an animal that has previously been immunised with the same antigen, induces an influx of antigen specific T cells into the lung airways. This is of course useful for immunotherapy of lung infections and lung cancer.

In particular, the invention provides a method for determining the effectiveness of pulmonary immunisation of a subject, the method comprising:

(a) measuring the expression of CXCR6 in a test sample of mononuclear cells obtained from the lung of the subject more than 7 days after pulmonary immunisation;

(b) comparing the expression measured in step (a) with a control value of CXCR6 expression obtained using a control sample of mononuclear cells taken from the lung of the subject before pulmonary immunisation and thereby determining whether or not the pulmonary immunisation was effective;

wherein an increased expression of CXCR6 in the test sample compared with the control value indicates that the pulmonary immunisation was effective in the subject.

The invention also provides:

a method for determining the effectiveness of pulmonary immunisation of a subject, the method comprising:

(a) measuring the level of production of CXCL16 by a test sample of mononuclear cells obtained from the lung of the subject after pulmonary immunisation;

(b) comparing the production measured in step (a) with a control value of production of CXCL16 obtained using a control sample of mononuclear cells taken from the lung of the subject before pulmonary immunisation and thereby determining whether or not the pulmonary

immunisation was effective;

wherein an increased production of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was effective in the subject;

(a) measuring the concentration of CXCL16 in a test sample of blood obtained from the subject after pulmonary immunisation;

(b) comparing the concentration measured in step (a) with a control value of CXCL16 concentration obtained using a control sample of blood taken from the subject before pulmonary immunisation and thereby determining whether or not the pulmonary immunisation was effective; wherein an increased concentration of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was effective in the subject;

a method for diagnosing a pulmonary infection or lung cancer in a subject, the method comprising:

(a) measuring the expression of CXCR6 or level of production of CXCL16 in a test sample of mononuclear cells obtained from the lung of the subject;

(b) comparing the expression or level of production measured in step (a) with a control value of CXCR6 expression or of level of production of CXCL16 obtained using a control sample of mononuclear cells taken from a subject without a pulmonary infection or without lung cancer and thereby determining whether or not the subject has a pulmonary infection or lung cancer; wherein an increased expression of CXCR6 or an increased level of production of CXCL16 in the test sample compared with the control value indicates that the subject has a pulmonary infection or lung cancer;

(a) measuring the concentration of CXCL16 in a test sample of blood obtained from the subject;

(b) comparing the concentration measured in step (a) with a control value of CXCL16 concentration obtained using a control sample of blood taken from a subject without a pulmonary infection or without lung cancer and thereby determining whether or not the subject has a pulmonary infection or lung cancer;

wherein an increased concentration of CXCL16 in the test sample compared with the control value indicates that the subject has a pulmonary infection or lung cancer;

method for screening a compound for its ability to treat or prevent a pulmonary infection or lung cancer, the method comprising:

(a) providing a test sample of mononuclear cells from the subject;

(b) measuring the expression of CXCR6 or production of CXCL16 by the test sample;

(c) incubating the test sample with the compound; and

(d) measuring the expression of CXCR6 or production of CXCL16 by the test sample in the presence of the compound and thereby determining whether or not the compound is able to treat or prevent a pulmonary infection or lung cancer; wherein an increase in the expression of CXCR6 or in the production of CXCL16 by the test sample in the presence of the compound indicates that the compound is able to treat or prevent a pulmonary infection or lung cancer;

a compound capable of treating or preventing a pulmonary infection or lung cancer identified using a method of the invention;

a method of treating or preventing a pulmonary infection or lung cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically or prophylactically effective amount of a compound of the invention;

a method for determining the effectiveness of a treatment of a pulmonary infection or lung cancer in a subject, the method comprising:

(a) measuring the expression of CXCR6 or level of production of CXCL16 in a test sample of mononuclear cells obtained from the lung of the subject after the treatment;

(b) comparing the expression or level of production measured in step (a) with a control value of CXCR6 expression or of level of production of CXCL16 obtained using a control sample of mononuclear cells taken from the subject before the treatment and thereby determining whether or not the treatment was effective;

wherein a decreased expression of CXCR6 or a decreased level of production of CXCL16 in the test sample compared with the control value indicates that the treatment was effective;

(a) measuring the concentration of CXCL16 in a test sample of blood obtained from the subject after the treatment;

(b) comparing the concentration measured in step (a) with a control value of CXCL16 concentration obtained using a control sample of blood taken from a subject before the treatment and thereby determining whether or not the treatment was effective;

wherein a decreased concentration of CXCL16 in the test sample compared with the control value indicates that the treatment was effective;

a method for determining the continued effectiveness of a treatment of a pulmonary infection or lung cancer in a subject, the method comprising:

(a) measuring the expression of CXCR6 or level of production of CXCL16 in a test sample of mononuclear cells obtained from the lung of the subject; (b) comparing the expression or level of production measured in step (a) with a control value of CXCR6 expression or of level of production of CXCL16 obtained using a control sample of mononuclear cells taken from the subject earlier during the treatment and thereby determining whether or not the treatment is continuing to be effective;

wherein a decreased expression of CXCR6 or a decreased level of production of CXCL16 in the test sample compared with the control value indicates that the treatment is continuing to be effective;

(b) comparing the concentration measured in step (a) with a control value of CXCL16 concentration obtained using a control sample of blood taken from a subject earlier during the treatment and thereby determining whether or not the treatment is continuing to be effective;

wherein a decreased concentration of CXCL16 in the test sample compared with the control value indicates that the treatment is continuing to be effective;

a vaccine composition comprising CXCL16 or a variant thereof and an antigen;

a fusion protein comprising CXCL16 or a variant thereof and an antigen;

a polynucleotide encoding a fusion protein of the invention;

a method for inducing a pulmonary mononuclear cell response against an antigen in a subject, the method comprising administering to the subject an immunologically effective amount of a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen, and thereby inducing a pulmonary mononuclear cell response against the antigen in the subject;

a method for inducing a pulmonary mononuclear cell response against an antigen in a subject, the method comprising (a) immunising the subject with the antigen and (b) administering to the immunised subject a therapeutically or prophylactically effective amount of a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen, and thereby inducing a pulmonary mononuclear cell response against the antigen in the subject; and

use of CXCL16 or a variant thereof as an adjuvant.

Brief Description of the Figures

Fig. 1 shows the expression of CXCR6 is upregulated on CD8 T cells in the lung after Ad85A i.n. immunisation. Mice were immunised with Ad85A by the i.n. or i.d. route. Five weeks later lymphocytes from various tissues/organs of (A) BALB/c and (B) C57BL/6 were co-stained for CD8 and CXCR6 expression. The data shown is representative of 2 independent experiments.

Fig. 2 shows CXCR6 expression is sustained on CD8 T cells in i.n. immunised Balb/c mice over time. The percentage of spleen and lung CD8 CXCR6⁺ from Ad85A i.n. and Ad85A i.d. mice at 7 weeks and 3 months post-immunisation are shown. The data are from one representative of 2-4 independent experiments.

Fig. 3 shows the numbers of CXCR6+ cells in BAL versus residual lung tissue. BALB/c mice were immunized i.n. or i.d. with Ad85A. Lymphocytes from BAL and residual lung tissue (processed after collection of BAL) from mice 3 weeks post-immunization, were stained for CD8, CXCR6 and Tetramer containing the dominant CD8 epitope of antigen 85A. Numbers of cells from BAL or residual lung of Ad85A i.d. (black bars) or i.n. (white bars) are shown, (A)

CD8+CXCR6+ and (B) CD8+Tetramer+CXCR6+ cells. The results are the mean of 3 ± SD mice from one of two independent experiments. Data from all repeats were analyzed by Kruskall-Wallis test followed by Dunn's Multiple comparison test. * indicates p-value <0.05 (Similar result with Mann- Whitney test) .

Fig. 4 shows that antigen specific lung cells express CXCR6. The dot plots show the percentage of CXCR6 Tetramer⁺ cells in the CD8 T cell population isolated from lungs of Balb/c mice immunised with Ad85A i.n. 7 weeks previously. The figures shown are representative of 2 independent experiments.

Fig. 5 shows that CXCR6⁺ cells from BAL of Ad85A i.n. immunised mice secrete IFNy. Lymphocytes were isolated from the BAL of mice immunised 13 weeks previously with Ad85A i.n. and stimulated with the dominant CD4 and dominant and subdominant CD8 peptides. The dot plot shows the percentage of IFNy⁺ cells in the CD8 CXCR6⁺ population in unstimulated and stimulated samples. The figures shown are representative of results obtained from 3-4 independent experiments.

Fig. 6 shows the phenotype of CD8 CXCR6⁺ lung cells following i.n. immunisation with Ad85A. Cells from the lungs of mice immunised 11 weeks previously with Ad85A intranasally were stained for CD27. The percentage of CD27 negative cells in the CXCR6⁺ or CXCR6^" populations is shown. Results are the mean ± SEM of 3 mice and are representative of results from two independent experiments.

Fig. 7 shows the expression of CXCR6 on lung T cells after immunization with rec85A by different routes. (A and B) BALB/c or C57BL/6 mice were immunized with rec85A s.c. or i.n. Four weeks after the final boost spleen and lung lymphocytes were isolated and (A) CD4 or (B) CD8 T cells stained for CXCR6. The results shown are the mean of 4 mice ± SD and are representative of results obtained from 2 independent experiments. (C and D) Expression of CXCR6 on lung and spleen T cells after immunization with ESAT6i_₂o peptide by different routes. C57BL/6 mice were immunized with ESAT6i_₂o peptide s.c. or i.n. Four weeks after the second immunization spleen and lung cells were stained for CD4, CD8 and CXCR6 and analysed by gating on CD4 (C) and CD8 (D) T cells. The results shown are the mean of 4 mice ± SEM and are representative of results obtained from 2 independent experiments. * indicates p<0.05 by Mann- Whitney test.

Fig. 8 shows the levels of CXCL16 protein in serum (A) and secreted by lung lymphocytes ex vivo (B) after immunisation. Serum was collected from mice six days after immunisation with Ad85A by the i.d. or the i.n. route. Lung lymphocytes were isolated 2 weeks post-immunisation and incubated for 6 hours at 37°C in RPMI+10%FCS without stimulation. The level of CXCL16 protein in serum and cell culture supernatant was measured by ELISA. The data shown are the mean concentration (±SD) of 5-6 mice per group (A) or 4-7 mice per group (B) from 2 independent experiments.

Fig. 9 shows the number of CD4⁺, CD8⁺ and CD8 Tetramer⁺ cells isolated from the BAL of Ad85A i.d. immunised mice after i.n. delivery of CXCL16 or rec85A proteins. Mice were immunised with Ad85A by the i.d. route. Three weeks post-immunisation rec85A or CXCL16 protein or a combination of both were administered i.n. Four day post-administration, BAL was collected from individual mice and the number of CD4⁺, CD8⁺ and CD8 Tetramer⁺ cells determined by flow cytometry. The figures show the mean absolute number of each cell population recovered (N=3, error bars show ±SD) from one of 2 independent experiments.

Fig. 10 shows that expression of CXCR6 is up-regulated on CD8⁺ T cells in the lung after Ad85A i.n. immunization. (A) BALB/c mice were immunized with Ad85A i.n. or i.d. Five weeks later CD8⁺ T cells from several tissues were analyzed for expression of CXCR6. The results shown are the mean values from 3 mice per group for lung, spleen and liver samples ± standard deviation (SD) while the facial lymph nodes and blood were pooled from the same mice for analysis and are representative of results obtained in two independent experiments. * indicates p = 0.03 by Mann- Whitney test of data from two experiments. B) the absolute numbers of CD8⁺ CXCR6⁺ cells in the lungs and spleen were also determined. The results shown are representative of two independent experiments.

Fig. 11 shows the expression of CXCR6 or IFNy on CD8⁺ cells in lungs or NALT.

BALB/c mice were immunized i.n with Ad85A in 50μ1 of PBS or 5μ1 with CT, to ensure that a strong NALT response was induced. Three weeks after immunization lung (Aand B) or NALT (C and D) CD8 T cells were analyzed for expression of IFNy (A, C) or CXCR6 (B, D). Figures for lungs are mean ± SEM for 4 mice. NALT cells were pooled from 4 mice.

Fig. 12 shows the properties of CXCR6⁺ cells in lungs after immunization. (A) Dot plots show the percentage of CXCR6⁺ Tetramer⁺ cells in the CD8⁺ gated T cell population isolated from lungs of BALB/c mice immunized with Ad85A i.n. or i.d. 7 weeks previously. (B) Histograms show mean ± SD from 2 experiments with 3 mice in each. (C) CD8⁺ CXCR6⁺ lung cells from mice immunized 11 weeks previously with Ad85A i.n. were stained for CD27. Histograms show mean ± SD of 3 mice and are representative of 2 experiments. (D) Lymphocytes from the BAL of mice immunized 13 weeks previously with Ad85A i.n. were stimulated with the dominant CD4 and dominant and subdominant CD8 peptides and stained for CD8, CXCR6 and intra-cellular IFNy. Histograms show mean ± SD of 3 mice and are representative of 4 independent experiments. * indicates p<0.05 by Mann- Whitney test.

Fig. 13 shows the number of CD4⁺, CD8⁺ and CD8⁺ Tetramer⁺ cells isolated from the BAL of Ad85A i.d. immunized mice after i.n. delivery of CXCL16 or rec85A proteins. (A) Mice were immunized with Ad85A i.d. Three weeks post-immunization rec85A, CXCL16 protein or both were administered i.n. Four days later, BAL was collected from individual mice and the number of CD4⁺, CD8⁺ and CD8⁺ Tetramer⁺ cells determined by flow cytometry. The figures show the mean number of each cell population recovered ± SD, from 3 mice in one of 2 independent experiments. (B) Mean mycobacterial burden in the lungs of Ad85A i.d. immunized mice given CXCL16 or rec85A proteins i.n. and subsequently challenged i.n. with a low dose of Mtb. Mice were immunized and rec85A, CXCL16 protein or both were administered i.n. as described above. Four days later, mice were challenged i.n. with 200 CFU of Mtb. Seven days post-challenge, the lungs were removed, homogenized and plated on Middlebrook agar plates to determine the

mycobacterial load. The figure shows the mean log CFU per lung of 3-5 mice per group from a single experiment. The data were analyzed using the Kruskal- Wallis test followed by Dunn's multiple comparison test; * or ** indicates p<0.05 compared to PBS or CXCL16 respectively.

Fig. 14 shows Mtb CFU in the lungs of na^'ive mice receiving CXCR6⁺ or CXCR6 depleted lymphocytes i.n. BALB/c mice were immunized with Ad85A i.n. Four weeks later lung lymphocytes were isolated and enriched for CD8⁺ T cells. These were then magnetic bead depleted of CXCR6⁺ cells. 5 x 10⁵ enriched CD8⁺ cells or CD8⁺ cells depleted of CXCR6⁺ cells were administered i.n. to na^'ive mice, which were challenged with Mtb one day later. Lungs were collected ΐ χ Mtb CFU enumeration 7 days later. *p<0.05, *** p<0.001 by one-way Anova with Tukey's post test

Description of the Sequences

SEQ ID NO: 1 shows the mRNA sequence encoding wild-type human CXCR6.

SEQ ID NO: 2 shows the amino acid sequence of wild-type human CXCR6.

SEQ ID NO: 3 shows the mRNA sequence encoding wild-type mouse CXCR6.

SEQ ID NO: 4 shows the amino acid sequence of wild-type mouse CXCR6.

SEQ ID NO: 5 shows the mRNA sequence encoding wild-type cow CXCR6.

SEQ ID NO: 6 shows the amino acid sequence of wild-type cow CXCR6.

SEQ ID NO: 7 shows the mRNA sequence encoding variant 1 of human CXCL16.

SEQ ID NO: 8 shows the amino acid sequence of variant 1 of human CXCL16.

SEQ ID NO: 9 shows the mRNA sequence encoding variant 2 of human CXCL16.

SEQ ID NO: 10 shows the amino acid sequence of variant 2 of human CXCL16.

SEQ ID NO: 1 1 shows the mRNA sequence encoding wild-type mouse CXCL16.

SEQ ID NO: 12 shows the amino acid sequence of wild-type mouse CXCL16. SEQ ID NO: 13 shows the mRNA sequence encoding variant 1 of the alternatively- spliced mouse CXCL16.

SEQ ID NO: 14 shows the amino acid sequence of variant 1 of the alternatively- spliced mouse CXCL16.

SEQ ID NO: 15 shows the mRNA sequence encoding variant 2 of the alternatively- spliced mouse CXCL16.

SEQ ID NO: 16 shows the amino acid sequence of variant 2 of the alternatively- spliced mouse CXCL16.

SEQ ID NO: 17 shows the mRNA sequence encoding wild-type cow CXCL16.

SEQ ID NO: 18 shows the amino acid sequence of wild-type cow CXCL16.

SEQ ID NOs: 19 to 21 show the peptides used in the Examples.

Detailed Description of the Invention

It is to be understood that different applications of the disclosed methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a lung" includes "lungs", reference to "a sample" includes two or more such samples, reference to "a subject" includes two or more such subjects, reference to "an infection" includes two or more such infections, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Methods for determining the effectiveness of pulmonary immunisation of a subject

The present invention provides methods for determining the effectiveness of pulmonary immunisation of a subject. The methods are carried out in vitro on samples of cells.

Pulmonary immunisation concerns immunisation of the lungs. This typically involves intranasal (i.n.) or intraoral (i.o.) immunisation or other method which ensures that the antigen reaches the lungs. It may involve immunisation with a recombinant antigen or a vector, such as a virus, containing an antigen. In the context of the invention, pulmonary immunisation is effective if it induces a mononuclear cell response, preferably a T cell response, to the relevant antigen in the lungs. In other words, pulmonary immunisation is effective if it induces mononuclear cell immunity, preferably T cell immunity, to the antigen in the lungs. Pulmonary immunisation is also typically effective if it reaches the deep lung. Immunisation is discussed in more detail below with reference to using CXCL16 as an adjuvant.

In a first embodiment, the expression of CXCR6 is measured in a test sample of mononuclear cells obtained from the lung of the subject more than 7 days after pulmonary immunisation. The subject, CXCR6, the test sample and mononuclear cells are discussed in more detail below. The method may comprise measuring the level of CXCR6 protein in the test sample. Methods for doing this are well-known in the art. Suitable methods include, but are not limited to, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Immunoprecipitation, Western blot analysis, fluorescence-activated cell sorting (FACS) analysis, an immunofluorescence assay and a light emission immunoassay. Alternatively, the method may comprise measuring the level of CXCR6 mRNA in the test sample. Methods for doing this are well-known in the art. Suitable methods include, but are not limited to, Northern blotting, reverse transcription quantitative polymerase chain reaction (RT-PCR followed with qPCR), "tag based" technologies, such as serial analysis of gene expression (SAGE), microarrays or high throughput sequencing.

In the first embodiment, the test sample of mononuclear cells is obtained from the lung of the subject more than 7 days after pulmonary immunisation. The test sample of cells may be obtained at any time after 7 days after pulmonary immunisation. For instance, the test sample may be taken at any time from 8 days to 1 year after immunisation. The test sample of mononuclear cells is preferably obtained at least 10 days, at least 3 weeks, at least 7 weeks, at least 3 months or at least 7 months after pulmonary immunisation. It is surprising that CXCR6 is expressed by lung T cells so long after pulmonary immunisation. Prior to the invention, it would have been predicted that any up-regulation of CXCR6 expression by lung T cells would be transient and only associated with the phase of entry of mononuclear cells into the lungs. It certainly could not be predicted that pulmonary immunisation with a recombinant protein would lead to prolonged expression of CXCR6 by lung T cells as is demonstrated in the Example. In the first embodiment, the expression of CXCR6 measured in the test sample is compared with a control value of CXCR6 expression obtained using a control sample of mononuclear cells taken from the lung of the subject before pulmonary immunisation. An increased expression of CXCR6 in the test sample compared with the control value indicates that the pulmonary immunisation was effective. An increased expression of CXCR6 in a test sample compared with the control value preferably indicates that pulmonary immunisation increased the number of mononuclear cells in the deep lung that are specific for the antigen against which the subject was immunised.

A decreased expression of CXCR6 in the test sample compared with the control value indicates that the pulmonary immunisation was not effective. Similarly, no change in the expression of CXCR6 in the test sample compared with the control value indicates that the pulmonary immunisation was not effective.

In the first embodiment, the control value is obtained using a control sample of

mononuclear cells obtained from the lung of the subject before pulmonary immunisation. The control value is obtained by measuring the expression of CXCR6 in the control sample of cells. The control value may be derived from more than one control sample. In order to allow an effective comparison, the control value has the same units as the expression from the test sample with which it is being compared. If the method of the invention involves measuring the level of CXCR6 protein, the control value typically concerns the level of CXCR6 protein in the control sample. Similarly, if the method of the invention involves measuring the level of CXCR6 mRNA, the control value typically concerns the level of CXCR6 mRNA in the control sample. A person skilled in the art is capable of obtaining such a value. The control value is typically obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value may be used for multiple repetitions of the method of the first embodiment. The control value is preferably obtained under the same conditions, such as cell number and method of measuring expression, under which the method of the first embodiment is carried out.

In a second embodiment, the level of production of CXCL16 is measured in a test sample of mononuclear cells obtained from the lung of the subject after pulmonary immunisation. The method may comprise measuring the level of CXCL16 protein secreted by the test sample. Alternatively, the method may comprise measuring the level of CXCL16 mRNA in the test sample. Methods for doing this are discussed above.

In the second embodiment, the test sample of mononuclear cells may be obtained from the lung of the subject at any time after pulmonary immunisation. For instance, the test sample may be taken at any time from 3 days to 1 year after immunisation. Suitable times are discussed above with reference to the first embodiment.

In the second embodiment, the production of CXCL16 measured in the test sample is compared with a control value of CXCL16 production obtained using a control sample of mononuclear cells taken from the lung of the subject before pulmonary immunisation. An increased production of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was effective. An increased production of CXCL16 in the test sample compared with the control value preferably indicates that pulmonary immunisation increased the number of T cells in the deep lung that are specific for the antigen against which the subject was immunised.

A decreased production of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was not effective. Similarly, no change in the production of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was not effective.

In the second embodiment, the control value is obtained using a control sample of mononuclear cells obtained from the lung of the subject before pulmonary immunisation. The control value is obtained by measuring the production of CXCL16 in the control sample of cells. The control value may be derived from more than one control sample. In order to allow an effective comparison, the control value has the same units as the production from the test sample with which it is being compared. If the method of the invention involves measuring the level of secretion of CXCL16 protein, the control value typically concerns the level of secretion of

CXCL16 protein by the control sample. Similarly, if the method of the invention involves measuring the level of CXCL16 mRNA, the control value typically concerns the level of CXCL16 mRNA in the control sample. A person skilled in the art is capable of obtaining such a value. The control value is typically obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value may be used for multiple repetitions of the method of the second embodiment. The control value is preferably obtained under the same conditions, such as cell number and method of measuring production, under which the method of the second embodiment is carried out.

In a third embodiment, the concentration of CXCL16 is measured in a test sample of blood obtained from the subject after pulmonary immunisation. The method typically comprises measuring the concentration of CXCL16 protein. However, CXCL16 mRNA can also be measured. Methods for doing this are discussed above.

In the third embodiment, the test sample may be obtained from the subject at any time after pulmonary immunisation. For instance, the test sample may be taken at any time up to 1 year after immunisation. Suitable times are discussed above with reference to the first embodiment. The test sample is preferably obtained less than 2 weeks after pulmonary immunisation, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 days after pulmonary immunisation.

In the third embodiment, the concentration of CXCL16 measured in the test sample is compared with a control value of CXCL16 concentration obtained using a control sample of blood taken from the subject before pulmonary immunisation. An increased concentration of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was effective. An increased concentration of CXCL16 in the test sample compared with the control value preferably indicates that pulmonary immunisation increased the number of T cells in the deep lung that are specific for the antigen against which the subject was immunised.

A decreased concentration of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was not effective. Similarly, no change in the concentration of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was not effective.

In the third embodiment, the control value is obtained using a control sample of blood obtained from the subject before pulmonary immunisation. The control value is obtained by measuring the concentration of CXCL16 in the control sample. The control value may be derived from more than one control sample. In order to allow an effective comparison, the control value has the same units as the concentration from the test sample with which it is being compared. If the method of the invention involves measuring the concentration of CXCL16 protein, the control value typically concerns the concentration of CXCL16 protein by the control sample. A person skilled in the art is capable of obtaining such a value. The control value is typically obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value may be used for multiple repetitions of the method of the third embodiment. The control value is preferably obtained under the same conditions, such as, blood volume, cell number and method of measuring concentration, under which the method of the third embodiment is carried out.

In the first to third embodiments, the subject has preferably been immunised against a pathogen against which T cells are protective. In the first to third embodiments, the subject has more preferably been immunised against a pathogen that infects the lungs and against which T cells are protective. The pathogen is preferably selected from Pseudorabies virus, Influenza virus, Swine fever virus, Mycoplasma hyopneumoniae, Mycobacterium bovis, Bovine respiratory syncytial virus, Bovine viral diarrhoea virus (BVDV), Pasteurella multocida, Respiratory syncytial virus (RSV), Parainfluenza, adenovirus, Mycobacterium tuberculosis, Measles virus, Rubella virus, Yersinia pestis, Bacillus anthracis and Francisella tularensis. These pathogens are discussed in more detail below. The subject has most preferably been immunised with 85 A, ESAT-6, CFP-10 or TB 10.4 from Mycobacterium tuberculosis or a nucleoprotein or matrix protein from an influenza virus.

Alternatively, in the first to third embodiments, the subject has preferably been immunised against lung cancer. Preferred lung cancers include, but are not limited to, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). The subject has preferably been immunised with an antigen selected from MAGE-A1, MAGE- A3, MAGE-B2 MAGE-C1, BAGE, GAGE, SSX-2, NY-ESO-1, K -LC-1, CEA, MUC-1, Sialyl Lewis A and Lewis Y, Her-2 and WT-1.

Methods for diagnosing a pulmonary infection in a subject

The present invention provides methods for diagnosing a pulmonary infection or lung cancer in a subject. The methods are carried out in vitro on samples of cells. The pulmonary infection may be caused by any of the pathogens discussed above. Preferred lung cancers are discussed above.

The subject is preferably at risk of or predisposed to a pulmonary infection or lung cancer. For instance, the method is suitable for screening subjects who have been exposed to one or more of the pathogens discussed above. The method is also suitable for screening smokers. The patient can be asymptomatic. In a first diagnostic embodiment, the expression of CXCR6 or level of production of CXCL16 is measured in a test sample of mononuclear cells obtained from the lung of the subject. The expression of CXCR6 or production of CXCL16 measured in the test sample is compared with a control value of CXCR6 expression or CXCL16 production obtained using a control sample of cells taken from the lung of a subject without a pulmonary infection or without lung cancer.

An increased expression of CXCR6 or level of production of CXCL16 in the test sample compared with the control value indicates that the subject has a pulmonary infection or lung cancer. A decreased expression of CXCR6 or level of production of CXCL16 in the test sample compared with the control value indicates that subject does not have a pulmonary infection or lung cancer. Similarly, no change in the expression of CXCR6 or level of production of CXCL16 in the test sample compared with the control value indicates that the patient does not have a pulmonary infection or lung cancer.

In the first diagnostic embodiment, the control value is obtained using a control sample of mononuclear cells obtained from the lung of a subject without a pulmonary infection or without lung cancer. The control value is obtained by measuring the expression of CXCR6 or level of production of CXCL16 in the control sample of cells as described above. The control value is preferably derived from one or more control samples from more than one, such as 10, 20 or 30 or more, subjects without a pulmonary infection or without lung cancer. The control value may be the mean of the values from more than one subject. In order to allow an effective comparison, the control value has the same units as the expression or level of production from the test sample with which it is being compared. If the method of the invention involves measuring CXCR6 or

CXCL16 protein, the control value typically concerns the level of CXCR6 or CXCL16 protein in the control sample. Similarly, if the method of the invention involves measuring the level of CXCR6 or CXCL16 mRNA, the control value typically concerns the level of CXCR6 or CXCL16 mRNA in the control sample. A person skilled in the art is capable of obtaining such a value. The control value is typically obtained separately from the method of the invention. For instance, the control value may be obtained beforehand and recorded, for instance on a computer. The control value may be used for multiple repetitions of the method of the first diagnostic embodiment. The control value is preferably obtained under the same conditions, such as cell number and method of measuring expression, under which the method of the first diagnostic embodiment is carried out. The control value is typically obtained using similar subjects to that being tested, in particular using subjects of the same species, sex and age.

In a second diagnostic embodiment, the concentration of CXCL16 is measured in a test sample of blood obtained from the subject. The concentration of CXCL16 measured in the test sample is compared with a control value of CXCL16 concentration obtained using a control sample of blood taken from a subject without a pulmonary infection or without lung cancer. An increased concentration of CXCL16 in the test sample compared with the control value indicates that the subject has a pulmonary infection or lung cancer. A decreased concentration of CXCL16 in the test sample compared with the control value indicates that the subject does not have a pulmonary infection or lung cancer. Similarly, no change in the concentration of CXCL16 in the test sample compared with the control value indicates that the subject does not have a pulmonary infection or lung cancer.

In the second diagnostic embodiment, the control value is obtained using a control sample of blood obtained from a subject without a pulmonary infection or without lung cancer. The control value is obtained by measuring the concentration of CXCR6 in the control sample of cells as described above. The control value is preferably obtained as described above with reference to the first diagnostic embodiment, except of course the concentration of CXCR6 in the control sample of blood is measured.

Drug screening

The present invention also provides methods for screening a compound for its ability to treat or prevent a pulmonary infection or lung cancer. The pulmonary infection may be caused by any of the pathogens discussed above. Preferred lung cancers are also discussed above.

Compounds screened in accordance with the invention are preferably able to increase the expression of CXCR6 or production of CXCL16 by mononuclear cells from the subject.

Compounds that increase the expression of CXCR6 or production of CXCL16 by mononuclear cells from the subject are suitable for treating or preventing a pulmonary infection or lung cancer because they will cause mononuclear cells to migrate to the lungs of the subject. In other words, such compounds will induce mononuclear cell immunity, preferably T cell immunity, in the lungs of the subject.

The method comprises providing a test sample of mononuclear cells from the subject, measuring the expression of CXCR6 or production of CXCL16 by the test sample, incubating the test sample with the compound and measuring the expression of CXCR6 or production of CXCL16 by the test sample in the presence of the compound and thereby determining whether or not the compound is able to treat or prevent a pulmonary infection or lung cancer. The mononuclear cells are preferably from the lung of the subject.

Suitable compounds to be screened include, but are not limited to, proteins,

polynucleotides, small molecules, natural products and lipophilic drugs. The cells in each sample may be incubated with the compound in any volume, for any length of time and at any temperature. Suitable volumes, times and temperatures include, but are not limited to, those discussed below.

An increase in the expression of CXCR6 or production of CXCL16 by the test sample in the presence of the compound indicates that the compound is able to treat or prevent a pulmonary infection or lung cancer. A decrease or no change in the expression of CXCR6 or production of CXCL16 by the test sample in the presence of the compound indicates that the compound is not able to treat or prevent a pulmonary infection or lung cancer.

Compounds and their use

The present invention also provides compounds capable of treating or preventing a pulmonary infection or lung cancer identified using a screening method of the invention. The compounds increase the expression of CXCR6 or production of CXCL16 in mononuclear cells from a subject.

The compounds that are identified in accordance with the invention may be used to treat or prevent a pulmonary infection or lung cancer. The pulmonary infection may be caused by any of the pathogens discussed above. Preferred lung cancers are discussed above. The present invention provides a method of treating or preventing a pulmonary infection or lung cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically or

prophylactically effective amount of a compound of the invention. The present invention also provides a compound of the invention for use in a method of treating or preventing a pulmonary infection or lung cancer in a subject in need thereof. The invention also provides use of a compound of the invention for the manufacture of a medicament for treating or preventing a pulmonary infection or lung cancer in a subject in need thereof. The compound can be administered to the subject in order to prevent the onset of one or more symptoms of the infection or cancer. In this embodiment, the subject can be asymptomatic. The subject may have been exposed to one or more of the pathogens mentioned above or may have a predisposition to the infection or cancer. For instance, the subject may be a smoker. A prophylactically effective amount of the compound is administered to such a subject. A

prophylactically effective amount is an amount which prevents the onset of one or more symptoms of the infection or cancer.

The compound can be administered to the subject in order to reduce the symptoms of the infection or cancer. A therapeutically effective amount of the compound is an amount effective to ameliorate one or more symptoms of the infection or cancer. Typically, such an amount increases the number of CXCR6-expressing mononuclear cells in the lungs of the subject. This can be confirmed as described in the Example.

The compound can be administered to the subject by any suitable means. The compound can be administered by enteral or parenteral routes such as via oral, buccal, anal, pulmonary, intravenous, intra-arterial, intramuscular, intraperitoneal, intraarticular, topical or other appropriate administration routes.

The formulation of any of the compounds will depend upon factors such as the nature of the compound and the disorder to be treated. The compound may be administered in a variety of dosage forms. It may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), parenterally, subcutaneously, intravenously, intramuscularly, intrasternally, transdermally or by infusion techniques. The compound may also be administered as a suppository. A physician will be able to determine the required route of administration for each particular patient.

Typically, the compound is formulated for use with a pharmaceutically acceptable carrier or diluent and this may be carried out using routine methods in the pharmaceutical art. The pharmaceutical carrier or diluent may be, for example, an isotonic solution. For example, solid oral forms may contain, together with the active compound, diluents, e.g. lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants, e.g. silica, talc, stearic acid, magnesium or calcium stearate, and/or polyethylene glycols; binding agents; e.g. starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone; disaggregating agents, e.g. starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dyestuffs; sweeteners; wetting agents, such as lecithin, polysorbates, laurylsulphates; and, in general, non-toxic and pharmacologically inactive substances used in pharmaceutical formulations. Such pharmaceutical preparations may be manufactured in known manner, for example, by means of mixing, granulating, tabletting, sugar-coating, or film coating processes.

Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carriers, for example, saccharose or saccharose with glycerine and/or mannitol and/or sorbitol.

Suspensions and emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, or polyvinyl alcohol. The suspensions or solutions for intramuscular injections may contain, together with the active compound, a pharmaceutically acceptable carrier, e.g. sterile water, olive oil, ethyl oleate, glycols, e.g. propylene glycol, and if desired, a suitable amount of lidocaine hydrochloride.

Solutions for intravenous or infusions may contain as carrier, for example, sterile water or preferably they may be in the form of sterile, aqueous, isotonic saline solutions.

For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%.

Oral formulations include such normally employed excipients as, for example,

pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%> to 95% of active ingredient, preferably 25% to 70%. Where the pharmaceutical composition is lyophilised, the lyophilised material may be reconstituted prior to administration, e.g. a suspension. Reconstitution is preferably effected in buffer.

Capsules, tablets and pills for oral administration to a patient may be provided

with an enteric coating comprising, for example, Eudragit "S", Eudragit "L", cellulose acetate, cellulose acetate phthalate or hydroxypropylmethyl cellulose.

Pharmaceutical compositions suitable for delivery by needleless injection, for example, transdermally, may also be used.

A therapeutically or prophylactically effective amount of the compound is administered. The dose may be determined according to various parameters, especially according to the compound used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. Again, a physician will be able to determine the required route of administration and dosage for any particular patient. A typical daily dose is from about 0.1 to 50mg per kg, preferably from about 0. lmg/kg to 1 Omg/kg of body weight, according to the activity of the specific inhibitor, the age, weight and conditions of the subject to be treated, the type and severity of the disease and the frequency and route of administration. Preferably, daily dosage levels are from 5mg to 2g.

Compounds identified in accordance with the invention may be polynucleotides. Preferably, the polynucleotide, such as R A or DNA, in particular DNA, is provided in the form of an expression vector, which may be expressed in the cells of the subject to be treated. The polynucleotides may be naked nucleotide sequences or be in combination with cationic lipids, polymers or targeting systems. The polynucleotides may be delivered by any available technique. For example, the polynucleotide may be introduced by needle injection, preferably intradermally, subcutaneously or intramuscularly. Alternatively, the polynucleotide may be delivered directly across the skin using a polynucleotide delivery device such as particle-mediated gene delivery. The polynucleotide may be administered topically to the skin, or to mucosal surfaces for example by intranasal, oral, intravaginal or intrarectal administration.

Uptake of polynucleotide constructs may be enhanced by several known transfection techniques, for example those including the use of transfection agents. Examples of these agents includes cationic agents, for example, calcium phosphate and DEAE-Dextran and lipofectants, for example, lipofectam and transfectam. The dosage of the polynucleotides to be administered can be altered. Typically the polynucleotide is administered in the range of lpg to lmg, preferably to lpg to l0μg polynucleotide for particle mediated gene delivery and l0μg to lmg for other routes.

Determining the effectiveness of a treatment

The present invention also provides methods for determining the effectiveness of a treatment of a pulmonary infection or lung cancer in a subject. The subject has typically been diagnosed with the infection or cancer and has been prescribed a treatment. The treatment may involve any of those discussed above or any standard treatment of pulmonary infections or lung cancers. Suitable treatments are known in the art. In a first embodiment, the expression of CXCR6 or level of production of CXCL16 is measured in a test sample of mononuclear cells obtained from the lung of the subject after the treatment. Methods for doing this are discussed above with reference to determining the effectiveness of pulmonary immunisation. The expression of CXCR6 or level of production of CXCL16 measured in the test sample is compared with a control value of CXCR6 expression or CXCL16 production obtained using a control sample of mononuclear cells taken from the lung of the subject before treatment. A decreased expression of CXCR6 or production of CXCL16 in the test sample compared with the control value indicates that the treatment was effective. An increased expression of CXCR6 or production of CXCL16 in the test sample compared with the control value indicates that the treatment was not effective. Similarly, no change in the expression of CXCR6 or production of CXCL16 in the test sample compared with the control value indicates that the treatment was not effective.

In the first embodiment, the control value is obtained using a control sample of mononuclear cells obtained from the lung of the subject after treatment. The control value is obtained as discussed above with reference to determining the effectiveness of pulmonary immunisation.

In a second embodiment, the concentration of CXCL16 is measured in a test sample of blood obtained from the subject after treatment. The method typically comprises measuring the concentration of CXCL16 protein. Methods for doing this are discussed above with reference to determining the effectiveness of pulmonary immunisation.

In the second embodiment, the concentration of CXCL16 measured in the test sample is compared with a control value of CXCL16 concentration obtained using a control sample of blood taken from the subject after treatment. A decreased concentration of CXCL16 in the test sample compared with the control value indicates that the treatment was effective. An increased concentration of CXCL16 in the test sample compared with the control value indicates that the treatment was not effective. Similarly, no change in the concentration of CXCL16 in the test sample compared with the control value indicates that the treatment was not effective.

In the second embodiment, the control value is obtained using a control sample of blood obtained from the subject before treatment. The control value is obtained as described above with reference to determining the effectiveness of pulmonary immunisation. Determining the continued effectiveness of a treatment

The present invention also provides methods for determining the continued effectiveness of a treatment of a pulmonary infection or lung cancer in a subject. The subject has typically been diagnosed with the infection or cancer and has been prescribed a treatment. The treatment may involve any of those discussed above. The first and second embodiments of these "continued" methods correspond to the first and second embodiments of determining the effectiveness of a treatment respectively, except that the control sample is taken from the subject earlier during the treatment than the test sample.

Subjects, CXCR6, CXCL16 and samples used in the methods of the invention

Subject

In any of the methods discussed above, the subject is may be any mammal that expresses CXCR6 and/or CXCL16. Typically, the subject is human. However, it may be non-human.

Suitable non-human animals include, but are not limited to, primates, such as marmosets or monkeys, commercially farmed animals, such as horses, cows, sheeps or pigs, and pets, such as dogs, cats, mice, rats, guinea pigs, ferrets, gerbils or hamsters. The subject is preferably human, a mouse, a chicken, pig, a cow or a sheep.

Table 1 summarises common pulmonary infections for each preferred species and the pathogens that cause them.

Table 1

Species Infections (pathogen)

Human Tuberculosis (Mycobacterium tuberculosis)

Influenza (influenza viruses)

Respiratory syncytial virus infection (RSV)

Measles (Measles virus)

German Measles (Rubella virus)

Plague (Yersinia pestis)

Anthrax (Bacillus anthracis)

Pig Pseudoraibies (Pseudorabies virus) Swine 'flu (Influenza viruses)

Swine fever (Swine fever virus)

Pneumonia {Mycoplasma hyopneumoniae)

Cow Bovine tuberculosis (Mycobacterium bovis)

Bovine respiratory syncytial virus infection (Bovine respiratory syncytial virus) Bovine viral diarrhoea virus infection (BVDV)

Pasteurellosis (Pasteur ella multocida)

Sheep Pasteurellosis (Pasteur ella multocida)

Respiratory syncytial virus infection (RSV)

Pneumonia (Mycoplasma, Parainfluenza and adenoviruses)

CXCR6

The methods described above may involve measuring the expression of CXCR6. The expression of CXCR6 protein or the level of CXCR6 mRNA may be measured. The CXCR6 protein is preferably SEQ ID NO: 2, 4 or 6 or a naturally occurring variant thereof. In various preferred embodiments:

the subject is a human and the CXCR6 protein comprises the sequence shown in SEQ ID NO: 2 or a naturally occurring variant thereof;

the subject is a mouse and the CXCR6 comprises the sequence shown in SEQ ID NO: 4 or a naturally occurring variant thereof; and

the subject is a cow and the CXCR6 comprises the sequence shown in SEQ ID NO: 6 or a naturally occurring variant thereof.

A naturally occurring variant is a variant of CXCR6 that is detectable in an organism of the relevant species. For instance, a naturally occurring variant of SEQ ID NO: 2 is a variant of SEQ ID NO: 2 that is detectable in a human subject. Naturally occurring variants will of course retain their ability to function as chemokine receptors.

Over the entire length of the amino acid sequence of SEQ ID NO: 2, 4 or 6, a naturally- occurring variant will preferably be at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2, 4 or 6. Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive -valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSP's containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The CXCR6 mRNA is preferably SEQ ID NOs 1 , 3 or 5 or a naturally occurring variant thereof. In various preferred embodiments:

the subject is a human and the CXCR6 mRNA comprises the sequence shown in SEQ ID NO: 1 or a naturally occurring variant thereof; the subject is a mouse and the CXCR6 mRNA comprises the sequence shown in SEQ ID NO: 3 or a naturally occurring variant thereof; and

the subject is a cow and the CXCR6 mRNA comprises the sequence shown in SEQ ID NO: 5 or a naturally occurring variant thereof.

A naturally occurring variant is a variant of CXCR6 mRNA that is detectable in an organism of the relevant species. For instance, a naturally occurring variant of SEQ ID NO: 1 is a variant of SEQ ID NO: 1 that is detectable in a human subject. Naturally occurring variants will of course retain their ability to encode chemokine receptors.

Over the entire length of the sequence of SEQ ID NO: 1, 3 or 5, a naturally occurring variant will preferably be at least 95%, 97% or 99% homologous based on nucleotide identity to the mRNA of SEQ ID NO: 1, 3 or 5. Homology can be determined as discussed above. Preferred naturally-occurring variants include:

nucleotides 82 to 1110 of SEQ ID NO: 1 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity;

nucleotides 75 to 1130 of SEQ ID NO: 3 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity; and

nucleotides 108 to 1130 of SEQ ID NO: 5 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity.

CXCL16

The methods described above may involve measuring the level of production of or concentration of CXCL16. The production or concentration of CXCL16 protein or mRNA may be measured. The CXCL16 protein is preferably SEQ ID NO: 8, 10, 12, 14, 16 or 18 or a naturally occurring variant thereof. In various preferred embodiments:

the subject is a human and the CXCL16 protein comprises the sequence shown in SEQ ID NO: 8 or 10 or a naturally occurring variant thereof;

the subject is a mouse and the CXCL16 comprises the sequence shown in SEQ ID NO: 12, 14 or 16 or a naturally occurring variant thereof; and

the subject is a cow and the CXCL16 comprises the sequence shown in SEQ ID NO: 18 or a naturally occurring variant thereof. A naturally occurring variant is a variant of CXCL16 that is detectable in an organism of the relevant species. For instance, a naturally occurring variant of SEQ ID NO: 8 is a variant of SEQ ID NO: 8 that is detectable in a human subject. Naturally occurring variants will of course retain their ability to function as chemokines.

Over the entire length of the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18, a naturally occurring variant will preferably be at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18. Homology may be determined as discussed above.

The CXCL16 mRNA is preferably SEQ ID NO: 7, 9, 11, 13, 15 or 17 or a naturally occurring variant thereof. In various preferred embodiments:

the subject is a human and the CXCL16mRNA comprises the sequence shown in SEQ ID NO: 7 or 9 or a naturally occurring variant thereof;

the subject is a mouse and the CXCL16 mRNA comprises the sequence shown in SEQ ID NO: 11, 13 or 15 or a naturally occurring variant thereof; and

the subject is a cow and the CXCL16 mRNA comprises the sequence shown in SEQ ID NO: 17 or a naturally occurring variant thereof.

A naturally occurring variant is a variant of CXCL16 mRNA that is detectable in an organism of the relevant species. It will of course retain its ability to encode a chemokine receptor. For instance, a naturally occurring variant of SEQ ID NO: 7 is a variant of that SEQ ID NO: 7 that is detectable in a human subject. Naturally occurring variants will of course retain their ability to encode chemokines.

Over the entire length of the sequence of SEQ ID NO: 7, 9, 11, 13, 15 or 17, a naturally- occurring variant will preferably be at least 95%, 97% or 99% homologous based on nucleotide identity to the mRNA of SEQ ID NO: 7, 9, 11, 13, 15 or 17. Homology can be determined as discussed above. Preferred naturally-occurring variants include:

nucleotides 533 to 1354 of SEQ ID NO: 7 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity;

nucleotides 533 to 1354 of SEQ ID NO: 9 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity;

nucleotides 538 to 1278 of SEQ ID NO: 11 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity; nucleotides 34 to 393 of SEQ ID NO: 13 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity;

nucleotides 34 to 393 of SEQ ID NO: 15 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity; and

nucleotides 24 to 782 of SEQ ID NO : 17 or a sequence having at least 95%, 97% or 99% homology thereto based on nucleotide identity.

Test and control samples

The methods of the invention may be carried out using any number of test samples. For instance, the method may be carried out using 1, 2, 5, 10, 15 or 20 or more test samples.

The relevant cells in the test samples are preferably captured or immobilized on a surface. Any method of immobilizing or capturing the cells can be used. The cells may be immobilized or captured on the surface using Fc receptors, capture antibodies, avidin:biotin, lectins, polymers or any other capture chemicals.

The test samples are typically present in wells. The samples are preferably present in the wells of a flat plate. The samples are more preferably present in the wells of a standard 96 or 384 well plate. Such plates are commercially available Fisher scientific, VWR suppliers, Nunc, Starstedt or Falcon. The cells are preferably immobilized or captured on a surface of one or more, preferably all, of the wells. Any method of immobilizing or capturing the cells can be used. The cells may be immobilized or captured on a surface of the well(s) by coating the surface with Fc receptors, capture antibodies, avidin:biotin, lectins, polymers or any other capture chemicals.

The wells typically have a capacity of from about 25μ1 to about 250μ1, from about 30μ1 to about 200μ1, from about 40μ1 to about 150μ1 or from about 50 to ΙΟΟμΙ.

Any number of cells may be present in each test or control sample. A person skilled in the art is able to determine an effective number of cells. The number is typically chosen on the basis of the number needed to form a confluent monolayer in the well within which the sample is contained. The numbers will differ between different cell types. Generally from about 10⁴ to about 10⁷ cells are present in each test or control sample. For example, from about 1.5 x 10⁴ to 7 x 10⁴ cells may be present in the sample. Preferably, about 2.0 x 10⁴ cells may be present in each sample.

In all the methods discussed above, the number of cells in each test sample and each control sample has preferably been calibrated. Such calibration does not need to be done every time that the methods are carried out. For instance, calibration can be done once and the methods may be carried out several times using the calibrated number of cells. In a preferred embodiment, the methods further comprise calibrating the number of cells. Calibration of the number of cells is intended to allow even small increases or decreases in the expression of CXCR6 or production of CXCL16 to be detected. Calibration of the number of cells therefore results in a sensitive assay.

The number of cells is preferably calibrated using several, such as 1, 2, 5, 6, 10, 15 or more, calibration samples of cells. Each calibration sample comprises a different number of cells within a range of numbers. This can be achieved by culturing the cells in each sample for different times before calibration is carried out. A person skilled in the art will be able to determine a suitable range of number of cells. For instance, if using a standard 96 well plate, a suitable range of number of cells 0 to 100,000 cells per well.

Mononuclear and T cells

In some of the methods discussed above, test and control samples of mononuclear cells obtained from the subject are used. Methods are known in the art for isolating and identifying mononuclear cells. One method is disclosed in the Example. The mononuclear cells preferably contain T cells. The T cells may be CD8⁺ or CD4⁺ cells.

Lung mononuclear cells may be obtained from a lung biopsy. Lung mononuclear cells are preferably obtained using Bronchoalveolar lavage (BAL). This involves inserting a bronchoscope into the lungs, using the bronchoscope to deliver fluid into a small part of the lung and then recollecting the fluid for examination. The BAL sample is typically processed prior to being used in the invention, for example by centrifugation, by passage through a membrane that filters out unwanted molecules or cells or by lysis of unwanted cells.

Generally, for drug screening, the mononuclear cells are taken from the individual in a blood sample, although other types of samples which contain mononuclear cells can be used. The sample may be added directly to the assay or may be processed first. Typically the processing may comprise standard techniques such as gradient centrifugation to separate the mononuclear cells, with resuspension in any suitable volume. Alternatively, the processing may comprise diluting of the sample, for example with water, buffer or media. The sample may be diluted from 1.5 to 100 fold, for example 2 to 50 or 5 to 10 fold. The processing may comprise separation of components of the sample. Typically mononuclear cells are separated from the samples. The mononuclear cells will comprise the T cells and antigen presenting cells (APCs). In another embodiment only T cells, such as only CD4⁺ or CD8⁺ T cells, can be purified from the sample. Mononuclear cells and T cells can be separated from the sample using techniques known in the art.

Preferably the mononuclear cells used in the assay are in the form of unprocessed or diluted samples, are freshly isolated T cells (such as in the form of freshly isolated mononuclear cells or PBMCs) which are used directly ex vivo, i.e. they are not cultured before being used in the method or are thawed cells (which were previously frozen). However the mononuclear cells can be cultured before use, for example in the presence of the antigen, and generally also exogenous growth promoting cytokines. During culturing the antigen is typically present on the surface of APCs, such as the APC used in the method. Pre-culturing of the mononuclear cells may lead to an increase in the sensitivity of the method. Thus the mononuclear cells can be converted into cell lines, such as short term cell lines using techniques known in the art.

Culturing of mononuclear cells allows equal numbers of cells to be present in each sample being assayed. Alternatively, if the cells are immobilized or captured, the mononuclear cells can be counted before plating. The importance equal number of cells is discussed above. Techniques for culturing mononuclear cells are well known to a person skilled in the art. The cells are typically cultured under standard conditions of 37°C, 5% C0₂ in medium supplemented with serum.

The cells may be cultured in any suitable flask or vessel. The cells may be cultured in wells of a flat plate, such as a standard 96 or 384 well plate.

Blood sample

In some embodiments, the test or control sample is a blood sample and is typically processed as described below before being used in the methods. The test or control sample may be blood, plasma or serum.

The sample is typically processed prior to being used in the invention, for example by centrifugation, by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells, or by lysis of unwanted cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be stored prior to use in the methods, preferably below -70°C, such as down to -200°C.

CXCL16 as an adjuvant

Vaccine compositions and fusion proteins

The inventors have surprisingly demonstrated that co-delivery of CXCL16 with antigen induces migration of large numbers of antigen-specific CD8 T cells into the lung. The invention therefore provides a vaccine composition comprising CXCL16 or a variant thereof and an antigen. As discussed in more detail below, the CXCL16 or variant thereof may be in the form of a protein or a polynucleotide. Similarly, the antigen may be in the form of a protein or a polynucleotide. As discussed in more detail below, the CXCL16 and antigen polynucleotides may be present in an expression cassette or a suitable vector.

The CXCL16 or variant thereof and the antigen are preferably present in a mixture (i.e. are not attached together). The mixture may be formulated in any of the ways discussed below. In another embodiment, the CXCL16 or variant thereof and antigen are covalently attached. The CXCL16 or variant thereof and antigen are covalently attached in such a way that the CXCL16 or variant thereof retains its ability to act as a chemokine and the antigen retains its ability to induce a mononuclear cell response, preferably a T cell response, in the subject. Any method of covalent attachment can be used. For instance, the CXCL16 or variant thereof and antigen may be attached using a bifunctional linker. Suitable linkers are known in the art. If the CXCL16 or variant thereof and antigen are both proteins, they may be covalently attached in the form of a fusion protein. In particular, the invention provides a fusion protein comprising CXCL16 or a variant thereof and an antigen. Methods for forming fusion proteins are well-known in the art. Typically, fusion proteins are expressed from a single polynucleotide sequence. The coding sequences of the CXCL16 or a variant thereof and antigen may be combined in any way to form a single polynucleotide sequence encoding the fusion protein. The invention therefore provides a polynucleotide encoding a fusion protein of the invention. The polynucleotide of the invention may be substantially isolated or part of an expression cassette or a suitable vector as discussed in more detail below.

Therapy The vaccine compositions, fusion proteins and polynucleotides of the invention may be used to induce a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against the antigen in a subject. In particular, the invention provides a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject, the method comprising administering to the subject an immunologically effective amount of a vaccine composition of the invention, a fusion protein of the invention or a

polynucleotide of the invention, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen, and thereby inducing a pulmonary mononuclear cell response against the antigen in the subject. An

immunologically effective amount is an amount which induces a pulmonary mononuclear cell response against the antigen in the subject.

The invention also provides:

a vaccine composition of the invention, a fusion protein of the invention or a

polynucleotide of the invention for use in a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen;

use of a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention for the manufacture of a medicament for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen;

a product containing (a) CXCL16 or variant thereof and (b) an antigen for simultaneous, separate or sequential use in a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against the antigen in a subject.

In other embodiments, the vaccine compositions, fusion proteins and polynucleotides of the invention may be used to induce a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against the antigen in a subject that has already been immunised with the antigen. In particular, the invention provides a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject, the method comprising (a) immunising the subject with the antigen and (b) administering to the immunised subject a immunologically effective amount of a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen, and thereby inducing a pulmonary mononuclear cell response against the antigen in the subject. An immunologically effective amount is an amount which induces a pulmonary mononuclear cell response against the antigen in the subject.

The invention also provides:

a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention for use in a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject, wherein the method comprises (a) immunising the subject with the antigen and (b) administering to the immunised subject an immunologically effective amount of the vaccine composition or the fusion protein, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen;

a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention for use in a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject that has been immunised with the antigen, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen;

use of a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention for the manufacture of a medicament for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject, wherein the medicament is used in a method comprising (a) immunising the subject with the antigen and (b) administering to the immunised subject the medicament, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen;

use of a vaccine composition of the invention, a fusion protein of the invention or a polynucleotide of the invention for the manufacture of a medicament for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against an antigen in a subject that has been immunised with the antigen, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen; a product containing (a) CXCL16 or variant thereof and (b) an antigen for simultaneous, separate or sequential use in a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against the antigen in a subject, wherein the method comprises (a) immunising the subject with the antigen and (b) administering to the immunised subject the CXCL16 or variant thereof and antigen; and

a product containing (a) CXCL16 or variant thereof and (b) an antigen for simultaneous, separate or sequential use in a method for inducing a pulmonary mononuclear cell response, preferably a pulmonary T cell response, against the antigen in a subject that has been immunised with the antigen.

In all of these embodiments, the pulmonary mononuclear cell response or pulmonary T cell response against the antigen is preferably for treating or preventing a pulmonary infection. In such embodiments, the invention results in a pulmonary mononuclear cell response against the pathogen causing the infection. The antigen is typically derived from the pathogen causing the infection. The pulmonary infection is preferably caused by any of the pathogens discussed above. Any of the antigens discussed above may be used.

Alternatively, in all of the embodiments discussed above, the pulmonary mononuclear cell response against the antigen is preferably for treating or preventing lung cancer. Preferred lung cancers include, but are not limited to, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), Suitable lung cancer antigens include, but are not limited to, MAGE-A1, MAGE- A3, MAGE-B2 MAGE-C1, BAGE, GAGE, SSX-2, NY-ESO-1, K -LC-1, CEA, MUC-1, Sialyl Lewis A and Lewis Y, Her-2 and WT-1.

CXCL16 or variant thereof

The CXCL16 or variant thereof may be in the form of a protein. The CXCL16 protein is preferably SEQ ID NO: 8, 10, 12, 14, 16 or 18 or a variant thereof. The specific protein or variant used will depend on the species of the subject. For instance, it is preferred that the CXCL16 protein is SEQ ID NO: 8 or 10 or variant thereof if the subject is a human. The specific sequences of CXCL16 for others species is discussed above.

A variant of SEQ ID NO: 8, 10, 12, 14, 16 or 18 is a protein that has an amino acid sequence which varies from that of SEQ ID NO: 8, 10, 12, 14, 16 or 18 and which retains its ability to act as a chemokine. The ability of a variant to act as a chemokine can be assayed using any method known in the art. For instance, the ability of the variant to attract CXCR6+ mononuclear cells can be assayed.

The variant may be a naturally occurring variant as discussed above or a non-naturally occurring variant produced by recombinant technology. Over the entire length of the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%) and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 200 or more, for example 300, 400, 500 or 600 or more, contiguous amino acids ("hard

homology"). Homology can be determined as discussed above.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 or more substitutions. Conservative substitutions may be made. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 2 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 3.

Table 2 - Chemical properties of amino acids

Ala aliphatic, hydrophobic, neutral Met hydrophobic, neutral

Cys polar, hydrophobic, neutral Asn polar, hydrophilic, neutral

Asp polar, hydrophilic, charged (-) Pro hydrophobic, neutral

Glu polar, hydrophilic, charged (-) Gin polar, hydrophilic, neutral Phe aromatic, hydrophobic, neutral Arg polar, hydrophilic, charged (+)

Gly aliphatic, neutral Ser polar, hydrophilic, neutral

His aromatic, polar, hydrophilic, Thr polar, hydrophilic, neutral

charged (+)

lie aliphatic, hydrophobic, neutral Val aliphatic, hydrophobic, neutral

Lys polar, hydrophilic, charged(+) Trp aromatic, hydrophobic, neutral

Leu aliphatic, hydrophobic, neutral Tyr aromatic, polar, hydrophobic

Table 3 - Hydropathy scale

Side Chain Hydropathy

He 4.5

Val 4.2

Leu 3.8

Phe 2.8

Cys 2.5

Met 1.9

Ala 1.8

Gly -0.4

Thr -0.7

Ser -0.8

Trp -0.9

Tyr -1.3

Pro -1.6

His -3.2

Glu -3.5

Gin -3.5

Asp -3.5

Asn -3.5

Lys -3.9

Arg -4.5

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 residues may be deleted, or more.

Variants may be fragments of SEQ ID NO: 8, 10, 12, 14, 16 or 18. Such fragments retain chemokine activity. Fragments may be at least 50, 100, 200, 250, 300, 350 or 400 amino acids in length. A fragment preferably comprises the domain of SEQ ID NO: 8, 10, 12, 14, 16 or 18 which interacts with CXCR6.

One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminus or carboxy terminus of the amino acid sequence of SEQ ID NO: 8, 10, 12, 14, 16 or 18 or a variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to 8, 10, 12, 14, 16 or 18 or a variant thereof.

The variant may be modified for example by the addition of histidine or aspartic acid residues to assist its identification or purification or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence.

The protein may be labelled with a revealing label. The revealing label may be any suitable label which allows the protein to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. ¹²⁵1, ³⁵S, ¹⁴C, enzymes, antibodies, antigens,

polynucleotides and ligands such as biotin.

The protein may be isolated from a chemokine producing organism, such as those discussed above, or made synthetically or by recombinant means. For example, the protein may be synthesised by in vitro translation and transcription. The amino acid sequence of the protein may be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When the protein is produced by synthetic means, such amino acids may be introduced during production. The protein may also be altered following either synthetic or recombinant production.

The protein may also be produced using D-amino acids. For instance, the protein may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins.

The protein may also contain other non-specific chemical modifications as long as they do not interfere with its ability to act as a chemokine. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH₄, amidination with methylacetimidate or acylation with acetic anhydride. The protein can be produced using standard methods known in the art. Polynucleotide sequences encoding a CXCL16 protein or variant thereof may be isolated and replicated using standard methods in the art. Chromosomal DNA may be extracted from a CXCL16 producing organism, such as those described above. The gene encoding the protein may be amplified using PCR involving specific primers. The amplified sequence may then be incorporated into a recombinant replicable vector such as a cloning vector. The vector may be used to replicate the polynucleotide in a compatible host cell. Thus polynucleotide sequences encoding the protein may be made by introducing a polynucleotide encoding the protein into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about replication of the vector. The vector may be recovered from the host cell.

The polynucleotide sequence may be cloned into a suitable expression vector. In an expression vector, the polynucleotide sequence encoding a protein is typically operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell. Such expression vectors can be used to express a protein.

The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence

"operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotide may be introduced into the vector.

The expression vector may then be introduced into a suitable host cell. Thus, a protein can be produced by inserting a polynucleotide sequence encoding a protein into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions which bring about expression of the polynucleotide sequence.

The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide sequence and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. A T7, trc, lac, ara or _L promoter is typically used.

The host cell typically expresses the protein at a high level. Host cells transformed with a polynucleotide sequence will be chosen to be compatible with the expression vector used to transform the cell. The host cell is typically bacterial and preferably E. coli. Any cell with a λ DE3 lysogen, for example C41 (DE3), BL21 (DE3), JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express a vector comprising the T7 promoter.

A protein may be produced in large scale following purification by any protein liquid

chromatography system from chemokine producing organisms or after recombinant expression as described below. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system.

The CXCL16 or variant thereof may be in the form of a polynucleotide. The

polynucleotide may be DNA, such as cDNA, or RNA, such as mRNA. The CXCL16 DNA is preferably a sequence which is complementary to SEQ ID NO: 7, 9, 11, 13, 15 or 17 or a variant thereof and comprises thymine (T) instead of uracil (U). The CXCL16 mRNA is preferably SEQ ID NO: 7, 9, 11, 13, 15 or 17 or a variant thereof. The specific polynucleotide or variant used will depend on the species of the subject. For instance, it is preferred that the CXCL16 mRNA is SEQ ID NO: 7 or 9 or a variant thereof if the subject is a human. The mRNA sequences of CXCL16 for others species are discussed above.

A variant of SEQ ID NO: 7, 9, 11, 13, 15 or 17 is a polynucleotide that has a sequence which varies from that of SEQ ID NO: 7, 9, 11, 13, 15 or 17 and which retains its ability to encode a chemokine. The ability of a variant to encode a chemokine can be assayed using any method known in the art. For instance, the polynucleotide can be expressed and the ability of its product to attract CXCR6+ T cells can be assayed.

The variant may be a naturally occurring variant as discussed above or a non-naturally occurring variant produced by recombinant technology. Over the entire length of the sequence of SEQ ID NO: 7, 9, 11, 13, 15 or 17, a variant will preferably be at least 50% homologous to that sequence based on nucleotide identity. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%>, 97%> or 99%> homologous based on nucleotide identity to the sequence of SEQ ID NO: 7, 9, 11, 13, 15 or 17 over the entire sequence. There may be at least 80%>, for example at least 85%>, 90%> or 95%>, nucleotide identity over a stretch of 600 or more, for example 900, 1200, 1500 or 1800 or more, contiguous nucleotides ("hard homology"). Homology can be determined as discussed above. A variant of SEQ ID NO: 1, 9, 11, 13, 15 or 17 may encode any of the protein variants of CXCL16 discussed above. Polynucleotides can be produced as discussed above.

The polynucleotide may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the protein in vivo in the subject. The expression cassette preferably also includes a polynucleotide encoding the antigen. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant viral vectors) which are suitable for use as reagents for

polynucleotide immunisation. Adenovirus vectors are particularly preferred. Such an expression cassette may be administered directly to the subject. Alternatively, a vector comprising a polynucleotide may be administered to the subject. Preferably the polynucleotide is prepared and/or administered using a genetic vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of the encoded protein.

Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a protein. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard we refer to Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

Preferably, the polynucleotide in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. "Operably linked" is defined above.

A number of expression systems have been described in the art, each of which typically consists of a vector containing a polynucleotide sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. The vectors may be for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. A "plasmid" is a vector in the form of an extrachromosomal genetic element. The vectors may contain one or more selectable marker genes, for example an ampicillin resistence gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. Vectors may be used in vitro, for example for the production of DNA or R A or used to transfect or transform a host cell, for example, a mammalian host cell. The vectors may also be adapted to be used in vivo, for example to allow in vivo expression of the polypeptide.

A "promoter" is a nucleotide sequence which initiates and regulates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term "promoter" or "control element" includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.

A polynucleotide, expression cassette or vector may additionally comprise a signal peptide sequence. The signal peptide sequence is generally inserted in operable linkage with the promoter such that the signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding sequence also in operable linkage with the promoter.

Typically a signal peptide sequence encodes a peptide of 10 to 30 amino acids for example 15 to 20 amino acids. Often the amino acids are predominantly hydrophobic. In a typical situation, a signal peptide targets a growing polypeptide chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved off in the endoplasmic reticulum, allowing for secretion of the polypeptide via the Golgi apparatus. Thus, a protein may be provided to an individual by expression from cells within the individual, and secretion from those cells.

Alternatively, polynucleotides may be expressed in a suitable manner to allow presentation of the encoded protein by an MHC class II molecule at the surface of an antigen presenting cell. For example, a polynucleotide, expression cassette or vector may be targeted to antigen presenting cells, or the expression of encoded protein may be preferentially stimulated or induced in such cells.

In some embodiments, the polynucleotide, expression cassette or vector will encode an adjuvant other than CXCL16, or an adjuvant other than CXCL16 will otherwise be provided. As used herein, the term "adjuvant" refers to any material or composition capable of specifically or non-specifically altering, enhancing, directing, redirecting, potentiating or initiating an antigen- specific immune response.

Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859 and 5,589,466. The polynucleotide can be introduced directly into the recipient subject, such as by standard intramuscular or intradermal injection; transdermal particle delivery;

inhalation; topically, or by oral, intranasal or mucosal modes of administration. The molecule alternatively can be introduced ex vivo into cells that have been removed from a subject. For example, a polynucleotide, expression cassette or vector may be introduced into APCs of an individual ex vivo. Cells containing the polynucleotides of interest are re-introduced into the subject such that an immune response can be mounted against the proteins encoded by the polynucleotides.

The proteins, polynucleotides, vectors or cells may be present in a substantially isolated form. They may be mixed with carriers or diluents which will not interfere with their intended use and still be regarded as substantially isolated. They may also be in a substantially purified form, in which case they will generally comprise at least 90%, e.g. at least 95%, 98%> or 99%, of the proteins, polynucleotides, cells or dry mass of the preparation.

Antigen

The antigen may be any antigen against which a pulmonary mononuclear cell response is desired. The antigen may be derived from any of the pathogens discussed above or from lung cancer. The antigen may be any of those discussed above. Preferred lung cancer antigens include, but are not limited to, MAGE-Al, MAGE -A3, MAGE-B2 MAGE-Cl, BAGE, GAGE, SSX-2, NY-ESO-1, K -LC-1, CEA, MUC-1, Sialyl Lewis A and Lewis Y, Her-2 and WT-1.

As discussed above, the antigen may be in the form of a protein or a polynucleotide. If the antigen is a polynucleotide, it is preferably in the same vector or expression cassette as the CXCL16 polynucleotide.

Formulations and compositions

The vaccine composition or fusion protein is preferably administered together with one or more pharmaceutically acceptable carriers or diluents and optionally one or more other therapeutic ingredients. The carrier (s) must be 'acceptable' in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Typically, carriers for injection, and the final formulation, are sterile and pyrogen free. Preferably, the carrier or diluent is thioglycerol or thioanisole.

Formulation of a suitable composition can be carried out using standard pharmaceutical formulation chemistries and methodologies all of which are readily available to the reasonably skilled artisan.

For example, a vaccine composition or fusion protein of the invention can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles and auxiliary substances are generally

pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, poly ethylenegly col, hyaluronic acid, glycerol, thioglycerol and ethanol. Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono-or di-glycerides.

Other parentally-administrable compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Alternatively, the vaccine composition or fusion protein of the invention may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.

The formulation of the vaccine composition or fusion protein of the invention will depend upon factors such as the nature of the substances in the composition/fusion protein and the method of delivery. The vaccine compositions and fusion proteins of the invention can be administered in a variety of dosage forms. They may be administered orally (e.g. as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules), topically, parenterally,

subcutaneously, by inhalation, intravenously, intramuscularly, intrasternally, transdermally, intradermally, sublingually, intranasally, buccally or by infusion techniques. The substance may also be administered as suppositories. A physician will be able to determine the required route of administration for each particular individual. The vaccine compositions and fusion proteins of the invention are preferably administered to the lungs of the subject. The vaccine compositions and fusion proteins of the invention are preferably administered by inhalation, intrasternally or intraorally.

The administered compositions will comprise a suitable concentration of the vaccine composition or fusion protein of the invention which is effective without causing adverse reaction. Typically, the concentration of each protein in the composition will be in the range of 0.03 to 200 nmol/ml. More preferably in the range of 0.3 to 200 nmol/ml, 3 to 180 nmol/ml, 10 to 150 nmol/ml, 50 to 200nmol/ml or 30 to 120 nmol/ml. The composition or formulations should have a purity of greater than 95% or 98% or a purity of at least 99%.

An adjuvant may also be used in combination with the vaccine composition or fusion protein. The adjuvant is preferably administered in an amount which is sufficient to augment the effect of the vaccine composition or fusion protein or vice versa. The adjuvant or other therapeutic agent may be an agent that potentiates the effects of the vaccine composition or fusion protein. For example, the other agent may be an immunomodulatory molecule or an adjuvant which enhances the response to the proteins or polynucleotides present in the vaccine composition or fusion protein.

In one embodiment, therefore, the vaccine compositions or fusion proteins are used for therapy in combination with one or more other therapeutic agents. The agents may be administered separately, simultaneously or sequentially. They may be administered in the same or different compositions as the vaccine composition or fusion protein. Accordingly, in a method of the invention, the subject may also be treated with a further therapeutic agent.

A composition may therefore be formulated with a vaccine composition or fusion protein of the invention and also one or more other therapeutic molecules. A vaccine composition or fusion protein of the invention may alternatively be used simultaneously, sequentially or separately with one or more other therapeutic compositions as part of a combined treatment.

Non-limiting examples of adjuvants include alum, monophosphoryl lipid, oligonucleotides, cholera toxin and Freund's complete or incomplete adjuvant.

Delivery methods

Once formulated the compositions or fusion proteins can be delivered to a subject in vivo using a variety of known routes and techniques. For example, the vaccine composition or fusion protein can be provided as an injectable solution, suspension, emulsion or dry powder and administered via parenteral, subcutaneous, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system, or using a patch. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, sublingual administration, and active or passive transdermal delivery techniques. Delivery to the lungs (pulmonary immunisation) is preferred.

Where a protein is to be administered, it is preferred to administer the protein to a site in the body where it will have the ability to contact suitable antigen presenting cells, and where it, or they, will have the opportunity to contact mononuclear cells of the individual. Where an APC is to be administered, it is preferred to administer the APC to a site in the body where it will have the ability to contact, and activate, suitable mononuclear cells of the individual.

Delivery regimes

Administration of vaccine composition or fusion protein may be by any suitable method as described above. Suitable amounts of the protein may be determined empirically, but typically are in the range given below. A single administration of each protein may be sufficient to have a beneficial effect for the patient, but it will be appreciated that it may be beneficial if the protein is administered more than once, in which case typical administration regimes may be, for example, once or twice a week for 2-4 weeks every 6 months, or once a day for a week every four to six months. As will be appreciated, each protein or polynucleotide, or combination of proteins and/or polynucleotides may be administered to a patient singly or in combination.

Dosages for administration will depend upon a number of factors including the nature of the composition, the route of administration and the schedule and timing of the administration regime. Suitable doses may be in the order of up to 15μg, up to 2(^g, up to 25μg, up to 3(^g, up to 5(^g, up to 10(^g, up to 500 μg or more per administration. Suitable doses may be less than 15μg, but at least lng, or at least 2ng, or at least 5ng, or at least 50ng, or least lOOng, or at least 500ng, or at least ^g, or at least 10μg. For some molecules, the dose used may be higher, for example, up to 1 mg, up to 2 mg, up to 3 mg, up to 4 mg, up to 5 mg or higher. Such doses may be provided in a liquid formulation, at a concentration suitable to allow an appropriate volume for administration by the selected route.

The following Example illustrates the invention.

Example Materials and Methods

Mice and immunisations

All experiments were performed with 6-8 week old female Balb/c or C57BL/6 mice (Harlan Orlac, Blackthorn, UK), were approved by the animal use ethical committee of Oxford University and fully complied with the relevant Home Office guidelines. Mice were immunised with a recombinant replication deficient adenovirus serotype 5 containing the 85 A antigen from M. tuberculosis (Ad85 A) (Ronan et al., PloS one 2009, 4(12):e8235). For intradermal (i.d.) immunisation mice were anesthetized and injected with 25 μΐ in each ear, containing a total of 2 x 10⁹ virus particles (v.p.) of Ad85A per mouse and for i.n. immunisation allowed slowly to inhale 50μ1 of 2 x 10⁹ v.p. of Ad85A.

Mice were also immunised with recombinant protein antigens of M. tuberculosis, rec85A or peptide encoding the first 20 amino acids of ESAT 6 (6 kDa early secretary antigenic target, ESAT6 i_2o. I.n. protein immunisations were conducted as above, delivering 2μg rec 85A protein or 20 μg of ESAT6i_2o mixed with 2 μg of cholera toxin (Sigma) into the nostrils. The rec85A or ESAT6i_₂o were also delivered by the subcutaneous route (s.c). 2μg rec 85 A protein or 20 μg of ESAT6i_₂o peptide were given with monophosphoryl lipid A Sigma adjuvant, system (Sigma) in 200μ1 according to the manufacturer's instructions. Protein immunisation was administered 3 times at 2 week intervals.

In some experiments mice were immunised with Ad85A by the i.d. route as above. 3 weeks post-immunisation recombinant CXCL16 at 2μg/mouse (R&D Systems) or rec85A at 2^g/mouse in 50μ1 PBS+0.5% BSA were delivered i.n. either singly or in combination to groups of immunised mice. 4 days after i.n. delivery, the mice were sacrificed and the BAL and lung tissue collected for analysis or the mice challenged with Mtb as described below.

Production of recombinant antigen 85A protein

Recombinant 85 A protein (rec85A) was produced as previously described (Franken et al, 2000. Purification of his-tagged proteins by immobilized chelate affinity chromatography: the benefits from the use of organic solvent. Protein Expr. Purif. 18:95-99). Briefly, the Mtb Rv3804c gene was amplified by PCR from genomic H37Rv DNA and cloned by Gateway technology (Invitrogen, Carlsbad, CA, USA) in a bacterial expression vector containing a histidine tag at the N-terminus. The protein was over-expressed in Escherichia coli BL21(DE3) and purified. Size and purity of rec85A was analyzed by gel electrophoresis and Western blotting with an anti-His antibody (Invitrogen, Carlsbad, CA, USA) and an anti-E. coli polyclonal antibody (Abeam, Cambridge, UK). Endotoxin content was below 50 IU/ mg recombinant protein as tested using a Limulus Amebocyte Lysate (LAL) assay (Cambrex, East Rutherford, NJ). Subsequently, the protein was tested in a lymphocyte stimulation assay to exclude antigen non-specific T-cell stimulation and cellular toxicity, using PBMC of in vitro PPD-negative healthy Dutch donors (Leyten et al., 2006. Human T-cell responses to 25 novel antigens encoded by genes of the dormancy regulon of Mycobacterium tuberculosis. Microbes Infect. 8:2052-2060).

Isolation of lymphocytes from bronchoalveo layer lavage (BAL), blood, lung, liver, facial lymph nodes, spleen and nasal associated lymphoid tissues (NALT)

Blood was collected from the jugular vein or by cardiac puncture into heparin tubes, diluted in PBS, then lymphocytes isolated by density centrifugation on Lymphoprep columns (Axis-Shield, Oslo, Norway). Cells from the interface were collected and washed. BAL was collected from lungs of mice by lung washes and pooled per group. The collected cells were centrifuged and resuspended in medium.

Lungs were perfused with PBS, cut into small pieces and digested with 0.7 mg/ml collagenase type I (Sigma, Poole, UK) and 30 mg/ml DNase I (Sigma) for 45 min at 37°C. Lung fragments were then crushed through a cell strainer using a 5 ml syringe plunger, washed, layered over Lympholyte (Cederlane, Ontario, Canada) and centrifuged at 1000 x g for 25 mins. Interface cells were collected and washed.

The spleens were passed through a cell strainer using a 5 ml syringe plunger, then red blood cells were lysed using RBC lysis buffer (Qiagen, Crawley, UK). The cells were washed.

Livers were perfused with PBS, then cut into small pieces and passed through a cell sieve. The single cell suspension was resuspended in 30% Percoll (GE Healthcare, Little Chalfont, UK) and layered over 70% Percoll. The gradient was centrifuged at 800 x g for 25mins and the interface cells were collected and washed.

Pooled parotid, mandibular and superficial cervical lymph nodes were collected and pooled from 3 mice per group. Single cell suspensions were made by crushing nodes between microscope slides. The cells were washed once.

Cells from the organised NALT (the paired lymphoid structures at the posterior end of the hard palate) were isolated as described in (Ronan et al, 2010. Nasal associated lymphoid tissue (NALT) contributes little to protection against aerosol challenge with Mycobacterium tuberculosis after immunisation with a recombinant adenoviral vaccine. Vaccine 28:5179-5184).

Isolated washed cells from all tissues/organs were resuspended in RPMI (Invitrogen) + 10%

FCS.

Purification and depletion of lymphocyte populations and cell transfer

CD8+ lung lymphocytes from mice immunized 4 weeks previously with Ad85A i.n. were enriched by first incubating with a cocktail of biotinylated monoclonal antibodies against different cell types (CD8 T cell isolation kit, Miltenyi Biotec, Surrey, UK), washing and labeling with anti-biotin microbeads, before separation on a LS magnetic column according to the manufacturer's instructions (Miltenyi). The flow though enriched CD8 T cells were divided into two. Half the cells were labeled with CXCR6-PE monoclonal antibody (Clone 221002, R&D Systems, Abingdon, UK) and the remainder were not. Both aliquots of cells were separately passed through LD magnetic columns. CD8 enriched cells contained -70% T cells and 3.2% of these were CXCR6+ while CXCR6 depletion reduced this to 1.3%. 5 xlO⁵ cells were administered i.n. in 50 ml PBS to each recipient mouse. The mice were challenged with Mtb the following day and organs harvested for enumeration of Mtb colony forming units (CFU) 7 days later.

Flow cytometry

Cells were cultured in RPMI supplemented with 10% heat-inactivated FCS, L-glutamine, penicillin and streptomycin for 6 hours. In some experiments, cells were stimulated with a mix of 3 peptides (Peptide Protein Research Ltd, Fareham, UK) encoding the dominant CD4 (Ag85A9₉_i i8aa TFLTSELPGWLQANRHVKPT (SEQ ID NO: 19)) and CD8 (Ag85A₇₀ vsaa MPVGGQSSF (SEQ ID NO: 20) and Ag85Ai₄₅-i52aa YAGAMSGL (SEQ ID NO: 21)) peptide epitopes (Ronan et al, 2009, supra). Each peptide was at a final concentration of 2μg/ml during the stimulation. After 2 hour at 37°C, Golgi Plug (BD Biosciences, Oxford, UK) was added according to the manufacturer's instruction and cells were incubated for an additional 4 hours before intracellular cytokine staining.

Cells were washed and incubated with CD16/CD32 mAB to block Fc binding. Subsequently the cells were stained for CD62L (MEL-14), CD127(A7R34), CD27(LG.7F9), CD19 (1D3), CD4 (RM4-5), IFNy (XMG1.2 and TNF (MP6-XT22) (eBioscience, Hatfield, UK), CXCR6 (221002) (R&D Systems, Abingdon, UK) and CD8 (53-6.7) (BD Bioscience) using the BD Cytofix/Cytoperm kit according to the manufacturer's instructions. In some experiments, cells were also stained with H- 2L^d 85A peptide ₇₀-₇₈ (MPVGGQSSF, SEQ ID NO: 21) tetramer (kindly provided by NIH Tetramer Facility). Cells were fixed with PBS + 1% paraformaldehyde, run on a LSRII (BD Biosciences) and analyzed using Flow Jo software (Tree Star Inc, Ashland, Oregon, USA). To obtain absolute cell numbers, 50 or 20μ1 of CountBright™ absolute counting beads suspension (Invitrogen) were added to each sample prior to analysis.

CXCL16 ELISA

Levels of CXCL16 protein were detected by ELISA (R&D Systems). Serum samples were collected by cardiac puncture. Lung lymphocytes isolated using the method described above were cultured in RPMI+10% FCS for 6 hours at 37°C without stimulation and the cell supernatant was assayed for CXCL16 production.

Infection with Mtb and determination of mycobacterial load

Mice were anaesthetized with isofluorane and infected i.n. wit Mtb (Erdman strain, kindly provided by Dr. Amy Yang, CBER/FDA) in 50μ1 PBS. Deposition in the lungs was measured 24 hours after challenge to determine the number of organisms deposited, which was of the order of 200 CFU. Mice were sacrificed at indicated times, the lungs were homogenized and the bacterial load determined by plating 10-fold serial dilutions of tissue homogenates on Middlebrook 7H11 agar plates (E&O Laboratories Ltd, Bonnybridge, UK). Colonies were counted after 3-4 weeks of incubation at 37°C in 5% C0₂.

Results

CXCR6 is upregulated on lung CD8 T cells after intranasal immunisation with Ad85A

BALB/c mice immunized with Ad85A i.n. but not i.d., show a statistically significant reduction in mycobacterial load following pulmonary Mtb challenge (Table 4).

Table 4. Lung mycobacterial load 6 weeks after aerosol challenge with 200 CFU of Mtb. Ad85A Immunization route Log CFU per lung (±SEM)^a

Not immunized 6.38 (±0.097) i.d. 6.40 (±0.077) i.n. 5.76 (±0.056) * ^aMean CFU from 5-6 mice per group challenged 4 weeks after immunization with Ad85A via indicated routes (26). The data was analyzed by Kruskal-Wallis test (overall p value 0.007) followed by Dunn's multiple comparison test, where * indicates p<0.05 compared to na^'ive or i.d. groups.

Since microarray analysis of CD8 T cells isolated from the lungs of mice immunized with Ad85A i.n. compared to Ad85A i.d., showed increased expression of CXCR6 (Lee et ah, 2010. Chemokine gene expression in lung CD8 T cells correlates with protective immunity in mice immunized intranasally with Adenovirus-85A. BMC Med. Genomics 3:46), we analyzed CXCR6 expression in lymphoid and non-lymphoid organs of mice immunized by the two routes. Figures 1 and 10 show that there were few CXCR6 CD8⁺ T cells in blood, spleen and pooled facial lymph nodes after Ad85A immunization i.n or i.d., with some up-regulation on liver CD8 T cells.

However, in the lung, only lymphocytes from mice immunized with Ad85A i.n. up-regulated CXCR6.

We have shown previously that in order to obtain protection against pulmonary Mtb challenge it is essential to use a 50μ1 i.n. inoculum to induce lower respiratory tract responses, while a 5μ1 inoculum induces a NALT response but no protection (Ronan et ah, 2010, supra). Here we analyzed CXCR6 expression after 5 or 50μ1 i.n. immunization with Ad85A. A 5μ1 non- protective inoculum did not induce CXCR6 expression on lung T cells (Figure 11), confirming that CXCR6 expression is a correlate of protective lower respiratory tract immunity.

CXCR6 expression on lung CD8 T cells is sustained after Ad85A i.n. immunisation

Effector CD8 T cells are typically short-lived and present during the course of an infection but after immunization with Ad85A i.n. antigen-specific lung T cells and protection against aerosol Mtb infection are sustained for several months (Ronan et al., 2009. supra). In accordance with this, although the number and percentage of lung antigen-specific CD8 T cells declined after a peak at 4-6 weeks post infection, we consistently demonstrated CD8+CXCR6+ antigen- specific cells in the lungs up to 3 months post-immunization (see, for example, Figures 2, 4 and 12) indicating that CXCR6 expression is long lived after i.n. immunization with Ad85A.

Furthermore, a small population (-1%) of CD8⁺ CXCR6⁺ cells was detected in lungs of 2 of 5 mice at 7 months post-immunisation (data not shown), thus indicting that CXCR6 expression is long lived after i.n. immunisation with Ad85A. This is in accordance with previous reports that the presence of antigen specific lung T cells and protection induced by Ad85A i.n. immunisation against aerosol M. tuberculosis infection is sustained for up to 7 months post-immunisation (Ronan et al., 2009, supra).

CD8 CXCR6⁺ cells are detected in the BAL only after immunisation with Ad85A i.n.

In mice infected with Mtb, the localization of effector T cells in BAL has been shown to correlate with protection induced by Ad85A i.n. immunization (Jeyanathan et al., 2010. Murine airway luminal antituberculosis memory CD8 T cells by mucosal immunization are maintained via antigen-driven in situ proliferation, independent of peripheral T cell recruitment. Am. J Resp. Crit. Care Med. 181 :862-872). To investigate if CXCR6 is expressed on these cells, BAL was collected from Ad85A i.n. and Ad85A i.d. mice 3 weeks post-immunization and the cells stained for CXCR6. Lymphocytes were also isolated from lung tissue after BAL collection to compare the number of CD8⁺CXCR6⁺ cells in these two compartments (Figures 3). Very few CD8⁺CXCR6⁺ cells are observed in the BAL of Ad85A i.d. mice, while they are abundant in BAL of Ad85A i.n. mice. Furthermore, in Ad85A i.n. immunized mice, a high proportion (-30%) of lung

CD8⁺CXCR6⁺ cells are in BAL. Double labeling of CD8 cells with CXCR6 and tetramer for the dominant CD8 H2^d-restricted epitope of antigen 85A also shows that many of the CXCR6⁺ are antigen- specific (Figures 2B and 12) suggesting that CXCR6 may be involved in targeting antigen- specific cells to the BAL-recoverable compartment.

Properties of lung CD8⁺CXCR6⁺ T cells

In order to analyze the properties of antigen-specific CXCR6⁺ cells, they were co-stained with tetramer for the dominant CD8 T cell epitope of 85A and antibodies to other functionally important molecules. Figures 4 and 12A and B show that 11 or 13 weeks post-immunization a high proportion of the CXCR6⁺ cells were tetramer positive (56%) and similarly many tetramer positive cells were CXCR6+ (27%). It is likely that some of the remaining CXCR6⁺ cells were adenovirus-specific, since lung cells from Ad85A i.n. immunized mice responded vigorously to a control adenovirus lacking 85A by cytokine production (data not shown). The CXCR6⁺ population was also stained with CD27 and expression of CD27 was down-regulated, indicating activation and recent exposure to antigen in vivo (Figure 3C).

BAL lymphocytes from Ad85 A i.n. immunized mice were stimulated with peptides representing the dominant CD4 and CD8 antigen 85 A T cell epitopes in the presence of brefeldin and co-stained for IFNy and CXCR6 to determine if CXCR6⁺ cells produce IFNy ex vivo. IFNy production was detected in a subset of CXCR6+ cells at various times post-immunization (Figure 12D), the percentage of CD8⁺CXCR6⁺IFNy⁺ varying from 14-36% of CXCR6⁺ cells. Stimulated CD8 CXCR6⁺ BAL cells also produced TNF detectable by intra-cytoplasmic staining (data not shown).

In summary, CXCR6⁺ cells induced after Ad85A i.n. immunization are highly activated, antigen- specific and capable of secreting IFNy and TNF. The cells appear to be fully functional and may contribute to the early control of Mtb that we have described previously in mice immunized with Ad85A i.n.

CXCR6 may serve as a marker of successful i.n. immunisation.

We next investigated if sustained up-regulation of CXCR6 in lung CD8 T cells was a response only to i.n. Ad85A. BALB/c or C57B1/6 mice were immunized with rec85A or ESAT6i_₂o peptide with adjuvants either i.n. or s.c. Four weeks after the last boost, the proportion of CXCR6⁺ cells was assessed. I.n. administration of rec85A or ESAT6 peptide induced expression of CXCR6 on lung T cells and in contrast to Ad85A i.n., protein or peptide immunization up-regulated CXCR6 predominantly on CD4⁺ T cells (Figure 7). CXCR6 is therefore a hallmark of successful i.n. immunization for both CD4 and CD8 cells irrespective of the nature of the immunogen.

CXCL16 levels are increased in serum and produced by lymphocytes after Ad85A i.n.

immunisation

Trafficking of cells into tissues occurs in response to a gradient of a chemokine ligand. CXCL16 is the only known ligand for CXCR6 and is usually expressed as a membrane bound molecule but an alternatively spliced, secreted form of the chemokine can be produced. Serum levels of CXCL16 were measured to determine if i.n. immunization elicited a CXCL16 gradient to recruit T cells from the circulation. Measurements were made 6 days and 2, 3 and 12 weeks post- immunization. At day 6, serum CXCL16 was significantly elevated in i.n. compared to i.d.

immunized mice (Figure 8 A) but not at later times (data not shown). As CXCL16 may be constitutively expressed in lung epithelium, we investigated whether lung lymphocytes isolated from naive, i.n or i.d. immunized mice differed in their ability to secrete CXCL16. Lung mononuclear cells isolated from i.n. immunized mice secrete CXCL16 protein up to 3 weeks after immunization (Figure 8B). Stimulation with the dominant CD4 and dominant and subdominant CD8 peptides for 6 hours does not alter the levels of CXCL16 produced by i.n. immunized lung lymphocytes, nor does it induce CXCL16 production in lung lymphocytes isolated from i.d.

immunized mice (data not shown). Therefore i.n. immunization increases serum CXCL16 shortly after immunization, possibly reflecting formation of a chemokine gradient to recruit activated CXCR6 expressing T cells into the lung, where they may be retained partly as a result of sustained expression of the CXCL16 by lung lymphocytes.

CXCL16 and antigen increase numbers of BAL-recoverable lymphocytes

In order to determine if CXCL16 on its own is able to increase the number of effector T cells in the BAL-recoverable compartment, BALB/c mice were immunized with Ad85A i.d. to induce a systemic CD8 T cell immune response. Three weeks later CXCL16 protein, rec85A or both were administered i.n. to the mice and four days later BAL cells were recovered and analyzed. There was no difference between the number of CD4⁺, CD8⁺ or Tet⁺ T cells in the BAL of mice given CXCL16 or control PBS (Figure 9 and 13 A). In contrast, delivery of rec85A protein i.n. increased the number of cells in the BAL, with a preferential increase in the CD8 and Tet⁺ T cell population but co-delivery of CXCL16 and rec85A caused a much larger increase in the numbers of lymphocytes in the BAL, indicating that CXCL16 and rec85A synergize. When identically treated groups of mice were infected with Mtb, only Ad85 A i.d. immunized mice which received both CXCL16 and antigen 85 A four days prior to challenge, were able to reduce significantly the lung mycobacterial load 7 days after challenge wit Mtb (Figure 13B). Therefore, exogenous CXCL16 in conjunction with cognate antigen leads to the presence of large numbers of CD8 and CD4 T cells in the BAL-recoverable compartment, where they can mediate protection against Mtb infection. Depletion of CXCR6⁺ cells decreases the protective efficacy of CD8⁺ T cells

BALB/c mice were immunized with Ad85A i.n. and 4 weeks later T cells were isolated from the lungs. After enrichment of CD8 T cells by magnetic bead depletion of other cell types, the CD8 enriched T cells were labeled with CXCR6 antibody and depleted by passage over a second magnetic column. Equal numbers (5 x 10⁵) of CD8⁺CXCR6⁺ and CD8⁺CXCR6 depleted cells were transferred i.n. into naive mice and the following day the mice were challenged with Mtb. Although both populations reduced Mtb CFU compared to na^'ive mice given no cells,

CD8⁺CXCR6⁺ had a significantly greater effect than CD8⁺CXCR6 depleted cells (Fig 14).

Discussion

We have previously shown that a striking effect of immunization with Ad85A i.n. is an early inhibition of lung mycobacterial growth following pulmonary challenge with Mtb, in contrast to the late inhibitory effects of BCG and other parenterally administered vaccines. We propose that i.n. boosting after BCG is effective because the growth of Mtb is inhibited both early and later after challenge and that early inhibition of Mtb growth is dependent on the preferential location of highly activated, antigen-specific effector cells secreting IFNy in the BAL-recoverable

compartment. Here we have shown that i.n. immunization with either a recombinant adenoviral vector or protein/peptides with CT as adjuvant, up-regulates CXCR6 on lung T cells and that CXCR6 expression persists on CD8 T cells for at least 3 months post-immunization. Immunization with recombinant adenovirus induces predominantly CD8 while protein/peptide immunization induces mainly CD4 antigen-specific T cells expressing CXCR6. CXCL16, the only known ligand for CXCR6, is also produced by lung mononuclear cells after i.n. immunization, perhaps as a consequence of IFNy and TNF production by effector T cells.

Expression of CXCR6 on lung CD8 T cells 7 days after i.n. immunization with rotavirus has been reported and several studies suggest that CXCR6+ cells are attracted toward and maintained in situ by the presence of antigen. For example, CXCR6⁺ cells migrate towards sites of acute bacterial infections and are found at sites of chronic inflammation, including the lung in chronic obstructive pulmonary disease. Prolonged production of CXCL16 may aid retention of CXCR6⁺ cells. It is thus likely that a combination of long-term presence of antigen, the expression of CXCR6 and the production of CXCL16 by lung mononuclear cells contribute towards long-term maintenance of an effector population following i.n. immunization. Furthermore, CXCR6⁺ cells are enriched in the BAL of Ad85A i.n. compared to i.d.immunized mice, suggesting that CXCL16 may play a role in their localization in the BAL-recoverable compartment, a suggestion supported by the synergistic effect of i.n. delivery of rec85A antigen and CXCL16. As it is known that BAL lymphocytes play an important role in protection against pulmonary growth of Mtb following challenge (Figure 14), the presence of a lung population of antigen-specific CXCR6⁺ cells is effectively a signature of local protective immunity after i.n. immunization. Expression of CXCR6 elsewhere does not correlate with protection, since Ad85A i.n. and i.d. immunized mice have equal numbers of CXCR6⁺ cells in the spleen but the former are well protected and the latter not (Figure 10).

We have previously investigated the role of the NALT in immunity to Mtb in mice. Here we targeted the NALT with Ad85A and CT in a small volume inoculum (5 μΐ) with the aim of ensuring a powerful NALT response. Even with the addition of CT, no CXCR6⁺ cells are detected in the lung although some are present in the NALT (Figure 11). In contrast a large volume inoculum of Ad85A (50 μΐ) with no adjuvant induces CXCR6⁺ cells in the lung (Figure 11) and protects from Mtb challenge. These data confirm the independence of NALT and lower respiratory tract immune responses in mice and support an association between the presence of CXCR6⁺ cells in the lung and local protective immunity following immunization.

CXCR6 expression is associated with development of protective lower respiratory tract immunity and with enrichment of CXCR6⁺ antigen-specific cells in BAL after i.n. immunization but CXCL16 has been reported previously to be poorly chemotactic. Therefore we tested the effect of administering recombinant CXCL16 i.n. to the airways of mice previously immunized i.d.

CXCL16 alone has little chemotactic effect (Figure 13). However, CXCL16 in combination with cognate antigen increased the numbers of T cells in the BAL and many of these were antigen- specific. CXCL16 is constitutively expressed by lung epithelial cells and is therefore already present in the lungs but normally expressed as a membrane bound molecule. Our results suggest that introducing only soluble (and thus potentially gradient-forming) CXCL16 into the airways has little effect but addition of cognate antigen has a synergistic effect with CXCL16. These data suggest two possibilities. First, presentation of antigen to T cells may make the cells more responsive to CXCL16 and promote migration into the BAL-recoverable compartment from the surrounding lung tissue or blood. Alternatively, CXCL16⁺ antigen may induce local proliferation in the BAL recoverable compartment. Preliminary data indicate a similar increase in numbers of BAL recoverable T cells after i.n. administration of CXCL16 with cognate antigen to mice previously immunized s.c. or i.m. with protein antigen plus adjuvant (data not shown).

In order to confirm that CXCR6⁺ cells in BAL are important for protection we isolated lymphocytes from the lungs of Ad85A i.n. immunized mice and transferred CD8⁺ T cells or CD8⁺ T cells depleted of CXCR6⁺ cells into the lungs of naive mice, then challenged the mice with Mtb. The CXCR6 depleted cells were significantly less protective than total CD8⁺ cells, Clearly and not surprisingly, since not all antigen-specific lung T cells express CXCR6, the depleted cells can also decrease the Mtb load. Nevertheless the data support the idea that CD8 CXCR6⁺ cells are important for local protection against pulmonary Mtb challenge. Both CXCR6⁺ and CXCR6^" cells may recruit innate effector cells and the exact mechanisms of early inhibition of Mtb growth in the lungs remain to be determined.

Successful prophylactic immunization against organisms that are normally combated by T cell immune responses has proved extremely difficult but recently it has been recognized that this may be because it is important to harness both systemic and local immunity at the portal of entry. In the case of Mtb, it has become clear in experimental models, that it is surprisingly difficult to improve the level of protection by boosting with parenterally administered subunit vaccines after BCG priming, although there are some exceptions. In contrast, i.n. immunization is highly effective, most likely because lung-resident activated T cells can inhibit growth of mycobacteria early after infectious challenge, in contrast to parenteral immunization, which only inhibits growth much later. It has also been shown that the location of the cells in the lung is critical and that BAL- recoverable cells are highly effective in mediating protection against Mtb. These data strongly support the idea that the geography of the immune response is important and that an optimal strategy for development of an Mtb vaccine should take into account the properties of three populations of antigen-specific cells, those in the BAL-recoverable compartment, those in the remaining lung tissues and those outside the lung in the immune system.

Our data suggest that expression of CXCR6 on lung T cells after i.n. immunization is a marker for local protective immunity to Mtb and that CXCR6 and CXCL16 play a role in promoting localization of T cells within the BAL-recoverable compartment. Manipulation of CXCR6-CXCL16 by i.n. immunization or co-delivery of CXCL16 and antigen after parenteral immunization, are strategies to recruit and establish both CD4 and CD8 antigen-specific effectors at the site of pulmonary pathogen entry, leading to quick and effective clearance upon infection.

Claims

1. A method for determining the effectiveness of pulmonary immunisation of a subject, the method comprising:

(b) comparing the expression measured in step (a) with a control value of CXCR6 expression obtained using a control sample of mononuclear cells taken from the lung of the subject before pulmonary immunisation and thereby determining whether or not the pulmonary

immunisation was effective;

2. A method according to claim 1 , wherein the test sample was obtained from the lung of the subject at least 3 weeks after pulmonary immunisation.

3. A method for determining the effectiveness of pulmonary immunisation of a subject, the method comprising:

immunisation was effective;

wherein an increased production of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was effective in the subject.

4. A method for determining the effectiveness of pulmonary immunisation of a subject, the method comprising:

(a) measuring the concentration of CXCL16 in a test sample of blood obtained from the subject after pulmonary immunisation; (b) comparing the concentration measured in step (a) with a control value of CXCL16 concentration obtained using a control sample of blood taken from the subject before pulmonary immunisation and thereby determining whether or not the pulmonary immunisation was effective; wherein an increased concentration of CXCL16 in the test sample compared with the control value indicates that the pulmonary immunisation was effective in the subject.

5. A method according to claim 4, wherein the test sample was obtained from the subject less than 2 weeks after pulmonary immunisation

6. A method according to any one of the preceding claims, wherein the subject was immunised against (a) a pathogen against which mononuclear cells are protective or (b) lung cancer.

7. A method according to claim 6, wherein the pathogen is selected from Pseudorabies virus, Influenza virus, Swine fever virus, Mycoplasma hyopneumoniae, Mycobacterium bovis, Bovine respiratory syncytial virus, Bovine viral diarrhoea virus (BVDV), Pasteurella multocida,

Respiratory syncytial virus (RSV), Parainfluenza, adenovirus, Mycobacterium tuberculosis, Measles virus, Rubella virus, Yersinia pestis, Bacillus anthracis and Francisella tularensis.

8. A method for diagnosing a pulmonary infection or lung cancer in a subject, the method comprising:

(b) comparing the expression or level of production measured in step (a) with a control value of CXCR6 expression or of level of production of CXCL16 obtained using a control sample of mononuclear cells taken from a subject without a pulmonary infection or without lung cancer and thereby determining whether or not the subject has a pulmonary infection or lung cancer; wherein an increased expression of CXCR6 or an increased level of production of CXCL16 in the test sample compared with the control value indicates that the subject has a pulmonary infection or lung cancer.

9. A method for diagnosing a pulmonary infection or lung cancer in a subject, the method comprising:

wherein an increased concentration of CXCL16 in the test sample compared with the control value indicates that the subject has a pulmonary infection or lung cancer.

10. A method for screening a compound for its ability to treat or prevent a pulmonary infection or lung cancer, the method comprising:

(a) providing a test sample of mononuclear cells from the subject;

(c) incubating the test sample with the compound; and

(d) measuring the expression of CXCR6 or production of CXCL16 by the test sample in the presence of the compound and thereby determining whether or not the compound is able to treat or prevent a pulmonary infection or lung cancer;

wherein an increase in the expression of CXCR6 or in the production of CXCL16 by the test sample in the presence of the compound indicates that the compound is able to treat or prevent a pulmonary infection or lung cancer.

11. A compound capable of treating or preventing a pulmonary infection or lung cancer identified using a method according to claim 10.

12. A method of treating or preventing a pulmonary infection or lung cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically or

prophylactically effective amount of a compound according to claim 11.

13. A method for determining the effectiveness of a treatment of a pulmonary infection or lung cancer in a subject, the method comprising:

wherein a decreased expression of CXCR6 or a decreased level of production of CXCL16 in the test sample compared with the control value indicates that the treatment was effective.

14. A method for determining the effectiveness of a treatment of a pulmonary infection or lung cancer in a subject, the method comprising:

wherein a decreased concentration of CXCL16 in the test sample compared with the control value indicates that the treatment was effective.

15. A method for determining the continued effectiveness of a treatment of a pulmonary infection or lung cancer in a subject, the method comprising:

(b) comparing the expression or level of production measured in step (a) with a control value of CXCR6 expression or of level of production of CXCL16 obtained using a control sample of mononuclear cells taken from the subject earlier during the treatment and thereby determining whether or not the treatment is continuing to be effective; wherein a decreased expression of CXCR6 or a decreased level of production of CXCL16 in the test sample compared with the control value indicates that the treatment is continuing to be effective.

16. A method for determining the continued effectiveness of a treatment of a pulmonary infection or lung cancer in a subject, the method comprising:

wherein a decreased concentration of CXCL16 in the test sample compared with the control value indicates that the treatment is continuing to be effective.

17. A method according to any one of claims 1, 8, 10, 13 or 15, wherein the subject is a human and the CXCR6 comprises the sequence shown in SEQ ID NO: 1 or 2 or a naturally occurring variant thereof.

18. A method according to any one of claims 1, 8, 10, 13 or 15, wherein the subject is a mouse and the CXCR6 comprises the sequence shown in SEQ ID NO: 3 or 4 or a naturally occurring variant thereof.

19. A method according to any one of claims 1, 8, 10, 13 or 15, wherein the subject is a cow and the CXCR6 comprises the sequence shown in SEQ ID NO: 5 or 6 or a naturally occurring variant thereof.

20. A method according to any one of claims 1, 8, 10, 13 or 15, wherein the subject is a human and the CXCL16 comprises the sequence shown in SEQ ID NO: 7, 8, 9 or 10 or a naturally occurring variant thereof.

21. A method according to any one of claims 3, 4, 8, 9, 10, 13, 14, 15 and 16, wherein the subject is a mouse and the CXCL16 comprises the sequence shown in SEQ ID NO: 11, 12, 13, 14, 15 or 16 or a naturally occurring variant thereof.

22. A method according to any one of claims 3, 4, 8, 9, 10, 13, 14, 15 and 16, wherein the subject is a cow and the CXCL16 comprises the sequence shown in SEQ ID NO: 17 or 18 or a naturally occurring variant thereof.

23. A vaccine composition comprising CXCL16 or a variant thereof and an antigen.

24. A vaccine composition according to claim 23, wherein the CXCL16 or a variant thereof and the antigen are mixed together, are covalently attached or form a fusion protein.

25. A fusion protein comprising CXCL16 or a variant thereof and an antigen.

26. A polynucleotide encoding a fusion protein according to claim 25.

27. A method for inducing a pulmonary mononuclear cell response against an antigen in a subject, the method comprising administering to the subject an immunologically effective amount of a vaccine composition according to claim 23 or 24, a fusion protein according to claim 25 or a polynucleotide according to claim 26, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen, and thereby inducing a pulmonary mononuclear cell response against the antigen in the subject.

28. A method for inducing a pulmonary mononuclear cell response against an antigen in a subject, the method comprising (a) immunising the subject with the antigen and (b) administering to the immunised subject a therapeutically or prophylactically effective amount of a vaccine composition according to 23 or 24, a fusion protein according to 25 or a polynucleotide according to claim 26, wherein the vaccine composition or fusion protein comprises the antigen or the polynucleotide encodes a fusion protein comprising the antigen, and thereby inducing a pulmonary mononuclear cell response against the antigen in the subject.

29. A method according to claim 28, wherein the method is for treating or preventing a pulmonary infection.

30. A method according to claim 29, wherein the infection is caused by pathogen against which T cells are protective.

31. A method according to claim 28, wherein the method is for treating or preventing lung cancer.

32. Use of CXCL16 or a variant thereof as an adjuvant.